CN101805744B - Methyl transferase gene in betaine synthesis path and modification and application thereof - Google Patents

Abstract

The invention discloses a methyl transferase gene in a betaine synthesis path and modification and application thereof. The method for synthesizing betaineb by using the methyl transferase gene comprises the following steps: colonizing glycine sarcosine N-methyl transferase gene (ApGSMT2) and dimethylglycine N-methyl transferase gene (ApDMT2) from aphanothece halophytica; generating ApGSMT2 and ApDMT2 genes by substituting codons which has lower occurring probability in higher plants in the ApGSMT2 and ApDMT2 with codons preferred by higher plants; and constructing series gene of a coding product, which are respectively positioned on a chloroplast and a mitochondria by using a method for fusing a protein positioning targeting sequence with the N-methyl transferase gene; the ApGSMT2 catalyzes glycine to be methylated by taking glycine and sarcosine as substrates; the ApDMT2 catalyzes dimethyl glycine to be methylated; the ApGSMT2 and the ApDMT2 carry out concerted catalysis to synthesize glycine betaine. ApGSMT2/ApDMT2 or modifier genes thereof are transferred under the synergism action into maize, wheat, cotton, soy beans, tobacco and other crops and high-concentration glycine betaine is accumulated in cells, so that the stress resistance of plants can be remarkably improved.

Description

Methyl transferase gene in a kind of trimethyl-glycine route of synthesis and modification and utilization

Technical field

The present invention disclose in a kind of cyanobacteria glycinebetaine route of synthesis two kinds of methyl transferase gene sequences with and the sequence of modifying factor and their encoded protein matter sequences respectively; And these two kinds of genes are used for the crop breeding for stress tolerance, belong to the genetically engineered field.Relate in particular to the clone and modify glycocoll sarkosine N-methyl transferase gene and N-methylsarcosine N-methyl transferase gene and the utilization of participating in the glycinebetaine biosynthetic pathway.

Background technology

In the natural synthetic plant of trimethyl-glycine; Glycinebetaine is as a kind of osmotic protection material; Can improve the resistance of plant to multiple abiotic stress (like saline and alkaline, arid, low temperature etc.), and closely related to the accumulating level of a certain resistance level of coercing and trimethyl-glycine.Genetically engineered research shows, in plant, accumulates glycinebetaine through transgenic technology and can improve its resistance.

At occurring in nature, the biosynthetic pathway of known glycinebetaine mainly contains two kinds, i.e. choline oxidative pathway and glycine methyl approach.

The choline oxidative pathway is substrate with the choline, through a step or the synthetic glycinebetaine of two-step oxidation reaction.This approach distributes extensively, by the choline dehydrogenase (CDH) and the betaine-aldehyde dehydrogenase catalysis that are present in mikrobe, plant and the Mammals.Be present in the E.C. 1.1.99.1 in the mikrobe, two successive reactions of ability catalysis synthesizing betaine.The catalysis glycinebetaine is synthetic successively for choline single oxygenase and betaine-aldehyde dehydrogenase in the higher plant of synthetic glycinebetaine.The enzyme gene of the synthetic glycinebetaine of existing a plurality of participation choline oxidation is cloned in succession.

The glycine methyl approach is a glycocoll through the continuous three step N-generation trimethyl-glycine that methylates; Be by glycocoll sarkosine methyltransgerase (the glycine sarcosine methyltransferase that relies on S-adenosylmethionine (SAM); GSMT) and rely on the sarkosine N-methylsarcosine methyltransgerase of SAM (sarcosine dimethylglycinemethyltransferase, SDMT) catalysis is accomplished respectively.From the eighties of last century later stage eighties, in some archeobacterias, eubacterium and extreme blue bacterium, found this approach successively, but understood seldom, only from a few species, clone so far and obtain genes involved.

The route of synthesis that with the choline is substrate is studied in multiple biology.Difference according to the choline mode of oxidizing in this approach relates to one or both enzymes.In higher plant, the research of trimethyl-glycine biosynthetic pathway mainly concentrates on chenopod, and research (McNeil et al., 1999,2001) is also arranged in Amaranthaceae and grass.In Chenopodiaceae and amaranthaceous plant, trimethyl-glycine is synthetic through the two-step oxidation reaction by choline, generates betaine aldehyde chloride by choline single oxygenase (CMO) catalysis earlier, and the latter generates trimethyl-glycine (Weretilnyk et al., 1989 under betaine-aldehyde dehydrogenase (BADH) catalysis; Akamoto, 2000).At present, CMO only finds in Chenopodiaceae and amaranthaceous plant, infers that other section plant possibly have different E.C. 1.1.99.1s.And the gene of coding BADH is cloned from spinach various plants such as (Weretilnyk and Hanson, 1990).BADH is not the enzyme of the synthetic special usefulness of glycinebetaine; It and omega-amino-SNDH (a kind of enzyme that in the polyamines metabolism, extensively exists) identical (

et al., 1994; Rathinasabapathi et al., 1994).

In intestinal bacteria, with the synthesis related reason bet operon coding of trimethyl-glycine (Andresen et al., 1988; Lamark et al., 1991): the betA CDH that encodes, the betB BADH that encodes; BetT coding choline transport albumen; The betI modulin of encoding, similar in transition pathway from the choline to the trimethyl-glycine and the plant, be enzymatic by two kinds; Be choline dehydrogenase (choline dehydrogenase, CDH) and BADH.CDH is that a kind of film that relies on oxygen combines flavoprotein, not only can be oxidized to betaine aldehyde chloride by the catalysis choline, and can be with the oxidation of much at one speed catalysis betaine aldehyde chloride.BADH is the soluble proteins of a kind of NAD of depending on, and betaine aldehyde chloride is had very high affinity (Landfald and Strom, 1986).

In mikrobe Arthrobacter globiformis (Arthrobacter globiformis) and nourishing Arthrobacter (A.pascens); The synthetic of glycinebetaine need a kind of enzyme be E.C. 1.1.99.1 (choline oxidase only; COD) catalysis (Ikuta et al., 1977) produces H simultaneously ₂O ₂

The approach that with the glycocoll is the substrate synthesizing betaine at first is to have a liking for (Nyyssola et al., 2000 of finding among mikrobe Actinopolysporahalophila and the Ectothiorhodospira halochloris of salt, 2001 at two kinds of utmost points; Waditee etal., 2003).In these mikrobes; Glycocoll is through the continuous three step N-generation glycinebetaines that methylate; The first step of reaction be by rely on S-adenosylmethionine (S-adenosylmethionine, glycocoll sarkosine methyltransgerase SAM) (glycine sarcosine methyltransferase, GSMT) catalytic; Second step of reaction is by sarkosine N-methylsarcosine methyltransgerase (the sarcosine dimethylglycine methyltransferase of GSMT or dependence SAM; SDMT) catalysis, final step are by SDMT catalytic (Nyyssola et al., 2001; Waditee et al., 2003).

The biosynthetic genetically engineered research of trimethyl-glycine

Biochemical analysis to natural accumulation trimethyl-glycine organism shows that trimethyl-glycine is stable in plant materials, and the rate-limiting step of trimethyl-glycine accumulation is its synthetic rather than its degraded (Rhodes and Hanson, 1993).Along with people's is understood in depth trimethyl-glycine biosynthetic pathway molecular mechanism in plant and the mikrobe, and a large amount of trimethyl-glycine synthetic genes involveds are also cloned in succession.This provides the molecule foundation in the organism that does not accumulate trimethyl-glycine, setting up the trimethyl-glycine biosynthetic pathway through engineered means.Participate in a plurality of genes of trimethyl-glycine synthetic such as COD, CDH, CMO, BADH etc. at present and in various plants, changed into merit, these transfer-gen plants have accumulated the trimethyl-glycine of different levels, have improved its resistance to multiple adverse circumstance.

In the higher plant; Choline is converted into trimethyl-glycine by choline MO (CMO) catalysis, successively from spinach (Rathinasabapathi et al., 1997), beet (Russell et al.; 1998) and prunella asiatica (Atriplex hortensis) (Shen Yiguo etc.; 2001) be cloned into CMO in and carried out transgenic research, these genes possess signal peptide sequence, are used to guide ripe peptide chain to be positioned stroma.Nuccio etc. (1998) change the CMO gene of spinach over to tobacco, and transgene product is positioned in the chloroplast(id), yet CMO is active and all very low (≤50nmol g of beet alkali content ^-1FW) (Nuccio et al., 2000).Yet when CMO expresses in tenuigenin, rather than when in chloroplast(id), expressing, beet alkali content improves (430nmol g greatly in the transgene tobacco ^-1FW).McNeil etc. (2000) use ¹⁴The metabolic kinetics of choline in the choline of C mark (trimethyl-glycine synthesizes substrate) the follow transgene tobacco finds that choline is trimethyl-glycine synthetic principal element (McNeil et al., 2000) in the restriction chloroplast(id) by kytoplasm to the transhipment of chloroplast(id).Phosphorylethanolamine-N-methyltransgerase (PEAMT) is the key enzyme in the choline route of synthesis, and its catalysis phosphorylethanolamine generates the methylation reaction of phosphorylcholine.McNeil etc. (2001) with the BADH gene cotransformation of the PEAMT of spinach and CMO gene and beet to tobacco chloroplast; Compare with single CMO genetic tobacco that changes; The free content of choline of changeing the polygene tobacco has improved 50 times, and beet alkali content has improved 30 times, is up to 1.8 μ mol g ^-1FW (McNeil et al., 2001).

From 1981 were separated to BADH from higher plant after, the research of relevant trimethyl-glycine synthetic enzyme had caused extensive concern very soon.From spinach (Weretilnyk and Hanson, 1990), beet (McCue and Hanson, 1992), Chinese sorghum (Wood et al., 1996), paddy rice (Nakamura et al., 1997), barley (Ishitani et al., 1995; Nakamura et al., 2001), be cloned into the BADH gene in prunella asiatica (Xiao Gang etc., 1995) and the prince's-feather (Legaria et al., 1998).In plants such as Chenopodiaceae, Amaranthaceae, BADH mainly is present in stroma; And BADH mainly is present in the peroxisome in monocotyledonss such as paddy rice, barley.The difference location of BADH in cell has the performance that is beneficial to its enzymic activity.Plant BADH gene expression amount is very low under the normal condition, some data (Nakamura et al., 1997; Xiao Gang etc., 1995; Weretilnyk et al., 1989) show that the BADH expression of gene receives Salt Stress-induced, but this reaction does not have salt inductive specificity, but to the reaction (Ishitani et al., 1995) of osmotic stress.

The research of changeing the BADH gene is more.Rathinasabapathi etc. (1994) will change tobacco at the BADH of spinach and beet gene the earliest, and transgene product is positioned in the chloroplast(id).The murder by poisoning that expression of exogenous gene render transgenic tobacco can be resisted betaine aldehyde chloride.When in substratum, adding the 5mM betaine aldehyde chloride, the content of trimethyl-glycine has reached 20 μ mol g in the transgene tobacco ^-1FW.Yet when in substratum, not adding betaine aldehyde chloride, transgene tobacco can not synthesizing betaine.Guo Yan etc. (1997) utilize particle bombardment with prunella asiatica BADH transgenosis in paddy rice, the transgenic paddy rice that filters out can be grown under salt stress and set seeds.Kumar etc. (2004) have obtained to change the Radix Dauci Sativae of BADH gene, and beet alkali content is respectively 93-101 μ mol g ^-1DW has improved the salt tolerance of transfer-gen plant greatly.Yang etc. (2005; 2007) will derive from the BADH gene transformation tobacco of spinach (Spinacia oleracea); Transfer-gen plant can synthesize glycinebetaine, and the synthetic trimethyl-glycine mainly accumulates in chloroplast(id), and transfer-gen plant has improved the resistance to high temperature stress.They think that under the moderate high temperature stress photosynthetic the keeping of transgene tobacco is owing to the provide protection of trimethyl-glycine to the Rubisco activating enzymes; Under serious high temperature stress, transgene tobacco has alleviated high temperature stress to the active oxygen injury that photosynthetic mechanism causes through improving the function of antioxidase system in the body, has kept the higher PS II active (Yang et al., 2007) of transgene tobacco.Existingly focus mostly in the conversion of individual gene about CMO and the engineered research of BADH, though the resistance of transgenic plant has raising in various degree, beet alkali content is very low.

Colibacillary CDH has the function of choline-monooxygenase (cmo) and betaine-aldehyde dehydrogenase concurrently, and this characteristic makes the genetically engineered that only shifts the betA gene can give the ability of plant accumulation glycinebetaine.1996, Lilius etc. changed the betA gene over to tobacco, and the salt tolerance of transgenic plant is compared with adjoining tree and is significantly improved, but they do not report the accumulating level of trimethyl-glycine.The betA gene that will comprise the plastosome signal for locating after Takabe etc. (1998) will modify imports paddy rice, and transgenic paddy rice can accumulate 5 μ mol g ^-1The trimethyl-glycine of FW, and its salt tolerant and drought resistance contrast be significantly improved.Wang Shufang etc. (2001) transfer to betA in the tomato, have improved the salt resistance of tomato.Under condition of salt stress, chlorophyll content of the Caulis et Folium Brassicae capitatae (Brassica oleracea var.capitata) of commentaries on classics betA gene and (Bhattacharya et al., 2004) that living weight all is significantly higher than the wild-type plant.We have obtained commentaries on classics betA gene corn and cotton through agrobacterium-mediated transformation in the laboratory; The resistance Physiological Analysis shows the cold-resistant and drought-resistant ability of transgenic corns (the Quan etal. that is significantly improved; 2004a; B), the drought resistance of transgene cotton also got tangible improvement the (Lv et al., 2007).

Synthetic of the intravital E.C. 1.1.99.1 of Arthrobacter (COD) catalysis trimethyl-glycine needs single enzyme, and therefore, the research of changeing the COD gene has more work.The COD enzyme is by codA (Arthrobactor globiformis) or cox (A.pascens) genes encoding, and using maximum at present is the codA gene.The codA gene has been changed over to Arabidopis thaliana (Hayashi et al., 1997,1998; Alia etal., 1998; Sakamoto et al., 2000), paddy rice (Akamoto et al., 1998; Mohanty et al., 2002) and in the tomato various plants such as (Park et al., 2004), the salt tolerant of transfer-gen plant (Hayashi et al., 1997,1998; Akamoto, 1998), cold-resistant (Park, 2004; Hayashi et al., 1997; Alia et al., 1998; Akamoto etal., 1998), heat-resisting (Alia et al., 1998) and freeze proof (Sakamoto et al., 2000) ability all is significantly increased.In Arabidopis thaliana, express the COD gene that has the chloroplast(id) signal for locating, trimethyl-glycine concentration is respectively 1.2 μ mol g in blade and the seed ^-1FW and 18 μ mol g ^-1DW (Hayashi et al., 1997), transfer-gen plant shows very strong salt tolerance.Coerce down freezing; The accumulation of GB in the transgenic arabidopsis plant significantly improved the survival rate of plant, and a lot of cool tone control gene (like cor6.6, cor15a; Cor47 and cor78) expression level compare no significant difference (Sakamoto etal., 2000) with wild-type.In the conversion of paddy rice, COD is positioned chloroplast(id), similar in trimethyl-glycine concentration and the transgenic arabidopsis in the transgenic paddy rice blade; If COD is positioned in the kytoplasm, synthetic trimethyl-glycine concentration ratio COD is positioned the high 3-5 of plant times (Akamoto et al., 1998) of chloroplast(id).This maybe be because choline concentration (choline synthesize the site) in tenuigenin compares in chloroplast(id) (Mudd and Datko, 1989 due to the height; Alia et al., 1999).Yet the accumulation of trimethyl-glycine in chloroplast(id) but can better protect PS II complex body to avoid the injury of high salt and low temperature stress (Akamoto et al., 1998).Change codA gene tomato and can accumulate 0.3-1.2 μ mol g ^-1The FW trimethyl-glycine, transfer-gen plant all is improved significantly in the resistance to cold from seed germination to blocky each stage.Under cold stress conditions, the rate ratio wild-type of transgenic Fructus Lycopersici esculenti has improved 10-30% (Park et al., 2004).Owing to be accompanied by H in the process of COD catalysis choline generation trimethyl-glycine ₂O ₂Generation, and the transfer-gen plant body in H ₂O ₂The suitable raising of level has activated the expression of some oxygen scavenging activity mechanism like Vitamin C oxidase and katalase etc., and these enzymes can be with H ₂O ₂Change into H ₂O, thus make H ₂O ₂Content maintains a level that lower pair cell is harmless.Simultaneously, transfer-gen plant activity in vivo oxygen removes that enzyme is active to be increased, and ability (Park et al., 2004) is coerced in the degeneration-resistant border that also helps improving plant.

Nyyssola etc. (2000) are respectively from two kinds of halophiles that greatly differ from each other in phylogenetics (Ectothiorhodospira halochloris and Actinopolyspora halophila; Promptly have a liking for the salt aphanothece) in be separated to two catalysis trimethyl-glycine synthetic methyltransgerase GSMT (glycocoll sarkosine methyltransgerase) and SDMT (sarkosine N-methylsarcosine methyltransgerase); Methyltransgerase sequence similarity on amino acid levels in two species is very high, and they all are that substrate passes through the synthetic glycinebetaine of two ground beetle glycosylation reactions with the glycocoll.Nyyssola etc. (2001) clone this two methyl transferase genes from E.halochloris, respectively called after EcGSMT and EcSDMT.Glycocoll N-methyltransgerase (GMT) catalysis glycine methyl changes into sarkosine in Mammals, and further methylation reaction does not take place.Lack homology (Waditee et al., 2003) between GMT in the Mammals and EcGSMT, EcSDMT and ApGSMT, the ApSDMT.2005; Waditee etc. arrive the gene cotransformation of GSMT that encodes among the A.halophila (ApGSMT) and SDMT (ApSDMT) in blue or green bacterium (Synechococcus sp.PCC 7942) of algae and the Arabidopis thaliana; Discovery is changeed the GB concentration of PCC 7942 cells of ApGSMT/ApDMT gene up to 200mM under salt stress; Content than the blue or green bacterium of the algae that changes bet (betA, betB) gene has improved 5 times, and the former can grow under the 600mMNaCl condition He in the seawater.The Arabidopis thaliana plant beet alkali content that changes ApGSMT/ApDMT significantly increases than the plant that changes the CMO gene, and supposition is owing to the substrate difference causes.In addition, GB has accumulation in the root that changes ApGSMT/ApDMT gene Arabidopis thaliana, stem, leaf and flower etc., and the salt tolerant of transfer-gen plant, cold-resistant comparing all with the Arabidopis thaliana of commentaries on classics CMO gene with drought resistance are significantly improved.Recently, Waditee etc. (2007) clone phosphoglycerate dehydrogenase gene (ApPGDH) from A.hanothece, and it is changed in intestinal bacteria E.coli and the Arabidopis thaliana.The result shows, no matter be in the intestinal bacteria of natural synthetic GB or can not the Arabidopis thaliana of self synthetic GB in, the heterogenous expression of ApPGDH has all improved the accumulating level of glycinebetaine.They are also with ApPGDH, ApGSMT and three genes of ApSDMT coexpression in Arabidopis thaliana; Under 100mM NaCl condition; The beet alkali content of transgenic arabidopsis root is 1.5 times (Waditee et al., 2007) of changeing ApGSMT/ApSDMT, and accumulating level reaches 1.6 μ molg ^-1FW.These results show, will be that the GB route of synthesis of substrate is introduced in the plant of not synthesizing GB with the glycocoll, are expected to make plant to accumulate a large amount of GB.

How improving cell glycinebetaine accumulating level, thereby further to improve transgenic plant resistance be one of problem of very paying close attention in the plant science field.Carrying out glycocoll abundant than choline in the genetically engineered cell capable of using of glycine methyl approach introducing is substrate, is expected to the glycinebetaine of accumulation high density in transfer-gen plant.

Term of the present invention and substratum

" stem apex " is meant ten to tens microns shoot apical meristem (shoot meristemetic) and greatly to tens millimeters bud-end (shoot tip), " shoot apical meristem " is meant apical growth awl and contiguous leaf primordium thereof.

In the present invention; The Transformation Program of agrobacterium tumefaciens (Agrobacterium tumefaciens) mediation is basically with (Ishida etc. such as Ishida; 1996) report that (acetosyringone As) waits phenolic cpd and neutral monose in transfection liquid, to add Syringylethanone.

In the present invention, the used Agrobacterium of transfection contains improved Ti-plasmid, latter's portability or do not carry herbicide resistance gene or other selective agent resistant gene.

In the present invention; The plant resistance is expressed and given to used herbicide resistance gene or other resistant gene as long as in transformed plant cells, enliven; The not restriction of the kind of the source of restriction gene, size, used promotor and institute's antiweed or other selective agent does not receive the restriction of agrobacterium strains yet.

In the present invention, minimum medium and plant growth regulating substance are used pressure sterilizing; Microbiotic isoreactivity composition is used filtration sterilization, behind medium sterilization, adds.

In the present invention, selective agent such as weedicide sprays with neutral tap water dissolving back.

In the present invention, the general employing of Agrobacterium cultivation YEP substratum (yeast extract 10g/l, peptone 10g/l, NaCl 5g/l, pH 7.0, solid medium adds 15g/l agar) cultivate.

The minimum medium that cereal crop seed germination and embryo culture are adopted is modified MS medium: KNO ₃1900mg/l, NH ₄NO ₃1650mg/l, CaCl ₂2H ₂O 440mg/l, MgSO ₄7H ₂O 370mg/l, KH ₂PO ₄H ₂O170mg/l, FeSO ₄7H ₂O 27.8mg/l, ZnSO ₄7H ₂O 10mg/l, MnSO ₄H ₂O 22mg/l, H ₃BO ₃10mg/l, Na ₂MoO ₄0.5mg/l, CuSO ₄5H ₂O 0.05mg/l, CoCl ₂2H ₂O 0.05mg/l, vitamin 10mg/l, pyridoxine hydrochloride 1mg/l, nicotinic acid 1mg/l, glycocoll 2mg/l, inositol 100mg/l, vitamin H 0.05mg/l, casein hydrolysate 250mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8-6.0.The kind of plant growth regulating substance and concentration are adjusted because of the genotype of cultivated material.

The minimum medium that leguminous crop seed germination and embryo culture are adopted is modified MS medium (the same) or improvement B5 medium.Medium component is planted in the back: KNO ₃2500mg/l, (NH ₄) ₂SO ₄134mg/l, CaCl ₂2H ₂O150mg/l, MgSO ₄7H ₂O 250mg/l, NaH ₂PO ₄H ₂O 150mg/l, FeSO ₄7H ₂O 27.8mg/l, ZnSO ₄7H ₂O 2mg/l, MnSO ₄H ₂O 10mg/l, H ₃BO ₃10mg/l, Na ₂MoO ₄0.5mg/l, CuSO ₄5H ₂O 0.05mg/l, CoCl ₂2H ₂O 0.05mg/l, vitamin 10mg/l, pyridoxine hydrochloride 1mg/l, nicotinic acid 1mg/l, glycocoll 2mg/l, inositol 100mg/l, vitamin H 0.05mg/l, casein hydrolysate 250mg/l, sucrose 20g/l, agar powder 7g/l, pH 5.8-6.0.The kind of plant growth regulating substance and concentration are adjusted because of the genotype of cultivated material.

In the present invention, transplanted seedling waters 1/2 modified MS medium inorganic salt solution or 1/2 improvement B5 medium inorganic salt solution, pH 6.0-7.0 every other day.

Summary of the invention

The purpose of this invention is to provide methyl transferase gene and modification and utilization in a kind of trimethyl-glycine route of synthesis to prior art.

The clone of ApGSMT2 gene and ApDMT2 gene

The present invention extracts and has a liking for salt aphanothece (Aphanothece halophytica) genomic dna; Conserved sequence design PCR primer according to mammals glycocoll N-methyltransgerase (GMT); Clone ApGSMT2, ApDMT2 gene; Make up plant expression vector, change over to and carry out the gene function checking in the tobacco.Through detecting the glycinebetaine content of transgene tobacco, in conjunction with gene sequencing relatively, confirmed that ApGSMT2 and ApDMT2 gene have function, be respectively the homologous gene of ApGSMT and ApSDMT.Concrete steps are:

30ml bacterium liquid is packed in the 50ml centrifuge tube, and 4 ℃ of centrifugal 10min of 5000g collect thalline.Resuspended 2 times of STE (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0)).In the 50ml centrifuge tube that fills thalline, add 20ml 2 * CTAB (100mmol/L Tris-HCl pH 8.0; 1.4mol/L NaCl; 20mmol/L EDTA pH8.0,2%CTAB) damping fluid, mixing; Incubation 90min in 65 ℃ of water-baths (adding 1% beta-mercaptoethanol before the preheating), the centre shakes up 2-3 time.Then centrifuge tube is taken out from water-bath, thing to be mixed adds equal-volume chloroform/primary isoamyl alcohol (24: 1) after being chilled to room temperature, puts upside down centrifuge tube gently several times, the centrifugal 10min of 10000rpm under the room temperature.Again supernatant is changed in the new 50ml centrifuge tube over to 0.8 times of volume isopropanol precipitating.Flocks changes over to and fills in the 70% alcoholic acid 7ml centrifuge tube washed twice.Discard ethanol, dry, 2ml TER (2ml TE+2 μ l 10mg/ml RNaseA) dissolving, 37 ℃ of water-bath hydrotropies.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), put upside down gently, make its abundant mixing extracting 10min, 4 ℃ of centrifugal 10min of 12000rpm.Get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24: 1), put upside down gently, make its abundant mixing extracting 10min, 4 ℃ of centrifugal 10min of 12000rpm.Get supernatant, add 1/10 volume 3M sodium-acetate (PH 5.2), behind the mixing, add 2 times of absolute ethyl alcohols that volume is cold.After mixing leaves standstill a little while gently, tick DNA with the rifle head, behind 70% alcohol flushing 2 times, vacuum-drying.TE dissolving DNA according to the DNA that carries of institute amount adding 100-500ul.

Retrieval GenBank DB is collected relevant methyl transferase gene, the corresponding protein sequence is analyzed design degenerated primer GSMT-F 5 ' ATGGCNAARAARGTNGA 3 ' and GSMT-R 5 ' YTARTCYTTY TTNGC3 '; DMT-F 5 ' ATGACNAARG CNGAYGC 3 ' and DMT-R 5 ' YTANGGYTTRTGRAA 3 '.Note: R:A/G; W:A/T; K:G/T; B:C/G/T; M:A/C; D:A/G/T; V:A/C/G; H:A/C/T; N:A/C/G/T; Y:C/T; S:G/C.

To have a liking for salt aphanothece genomic dna is template, uses degenerated primer GSMT-F and GSMT-R respectively, DMT-F and DMT-R, and amplification obtains dna fragmentation.Response procedures is 95 ℃ of preparatory sex change 5min; 95 ℃ of sex change 1min, 45 ℃ of annealing 1min, 72 ℃ are extended 30s, circulate 35 times; 72 ℃ are extended 7min.Adopt Takara to reclaim Kit and reclaim the dna fragmentation that pcr amplification obtains; Select

Easy Vector Systems Kit (Promega for use; USA) the PCR fragment is cloned, send order-checking company to check order.

Sequencing result shows, has obtained two long nucleotide sequences for 798bp and 834bp.Sequence to obtaining is analyzed, and has confirmed their two ORF that encode respectively.The protein sequence of two ORF sequence encodings is carried out the blast analysis, find them and report that the GSMT and the DMT sequence that derive from other kinds biology have very high similarity.They all contain characteristic motif (the Motif I of methyltransgerase further analysis revealed; Post I, Motif II and Motif III); Tentatively inferring them is the newcomers that have a liking for salt aphanothece methyl transferase gene, respectively called after ApGSMT2, ApDMT2 gene.

The present invention clone's the nucleotide sequence of having a liking for salt aphanothece glycocoll sarkosine N-methyl transferase gene (ApGSMT2) is shown in sequence table SEQ ID NO.5.

The ApGSMT2 amino acid sequence coded is shown in sequence table SEQ ID NO.6.

The present invention clone's the nucleotide sequence of having a liking for salt aphanothece N-methylsarcosine N-methyl transferase gene (ApDMT2) is shown in sequence table SEQ ID NO.7.

The ApDMT2 amino acid sequence coded is shown in sequence table SEQ ID NO.8.

The modification of ApGSMT2 gene and ApDMT2 gene

The present invention can efficiently express in cereal crop in order to make the gene of having a liking for salt aphanothece (a kind of blue-green algae); We select for use the preference codon of cereal plants and corn to replace the codon of part A pGSMT2 and ApDMT2; ApGSMT2 called after ApGSMT2d after the modification, the ApDMT2 unnamed gene after the modification is ApDMT2d.ApGSMT2d and ApDMT2d gene synthetic, and through sequence verification.

The nucleotide sequence of ApGSMT2d is shown in sequence table SEQ ID NO.9.

The nucleotide sequence of ApDMT2d is shown in sequence table SEQ ID NO.10.

Methylase and chloroplast protein are led peptide sequence and are merged

The present invention can effectively improve the resistance of plant in order to make ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d gene in higher plant cell; From realizing that glycinebetaine is in intracellular compartmentation accumulation angle; ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d are merged with peptide (transit peptide) coding region of leading of location chloroplast protein respectively; ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d albumen are positioned in the chloroplast(id) after synthetic, promote a large amount of glycinebetaine of accumulation in the chloroplast(id).Clone Arabidopis thaliana the RUBISCO small ylidene gene lead peptide-coding region, respectively with ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d gene fusion, produce ApTpGSMT2 and ApTpGSMT2d and ApTpDMT2 and ApTpDMT2d gene.

The RUBISCO small ylidene gene of Arabidopis thaliana lead peptide-coding region shown in sequence table SEQ ID NO.11.

Its coding lead peptide shown in sequence table SEQ ID NO.12.

Fusion rotein ApTpGSMT2 that the present invention produces and ApTpGSMT2d aminoacid sequence are shown in sequence table SEQ IDNO.13.

Fusion rotein ApTpDMT2 that the present invention produces and the aminoacid sequence of ApTpDMT2d are shown in sequence table SEQ IDNO.14.

Clone's methylase gene and mitochondrial protein are led peptide sequence and are merged

Gene fusion construct of the present invention accumulates the purpose of the resistance that reaches further raising transgenic plant to realize glycinebetaine in plant mitochondria.Plastosome is the main place in cellular energy source as ATP synthetic place in the cell, realizes that trimethyl-glycine helps improving the resistance of transgenic plant in Intramitochondrial accumulation.As with corn citrate synthase gene lead peptide-coding sequence and ApGSMT2 and ApDMT2 gene fusion, produce ApTpGSMT2m and ApTpDMT2m gene.

The corn mitochondrial protein is led peptide sequence shown in sequence table SEQ ID NO.15.

The fusion rotein ApTpGSMT2m sequence that the present invention produces is shown in sequence table SEQ ID NO.16.

The fusion rotein ApTpDMT2m sequence that the present invention produces is shown in sequence table SEQ ID NO.17.

The structure of plant expression vector

The present invention links to each other ApGSMT2/ApDMT, ApTpGSMT2d/ApTpDMT2d, ApTpGSMT2m/ApTpDMT2m etc. respectively with higher plant promotor such as cauliflower mosaic virus (CAMV) 35SRNA gene promoter, corn Ubiquitin gene promoter, Arabidopis thaliana RD29 promotor and gene 3 ' tail district; The fusion gene that formation efficiently expresses; And then construct the plant expression vector that carries ApGSMT2 and ApDMT, ApTpGSMT2d and ApTpDMT2d and ApTpGSMT2m and ApTpDMT2m respectively (target gene is started by different promotors respectively, and various combination can be arranged).As utilize mini-Ti plasmid generation T-DNA district to be different plant expression vectors shown in Figure 1.Conventional Protocols in Molecular Biology is adopted in recombination.

Gene function is confirmed and the assessment of using value

The present invention is in order to confirm whether ApGSMT2 and ApDMT2 have the catalytic methylation reactive activity; Can the two coordinate expression improve the content of cell glycinebetaine; ApGSMT2 and ApDMT2 are recombinated on the prokaryotic expression carrier; Change intestinal bacteria over to, measure the glycinebetaine content of transgenic intestinal bacteria and wild-type e. coli.Glycinebetaine extracts Xing and Rajashekar (2001) method of adopting, and content is used ¹H NMR measures (Bruker AM500NMR spectrometer).The content of glycinebetaine is than (in the LB nutrient solution) about the transgenic control cells does not double in e. coli bl21 (DE3) plysS of ApGSMT2 and ApDMT2 coexpression cell; In nutrient solution, increase 0.3-0.5mol/LNaCl; The glycinebetaine content ratio of transgenic cell not transgenic contrast increases 30%-60%; Transgenic cell shows than illumination and shows the salt tolerance that improves, and growth velocity is significantly higher than contrast.Be that ApGSMT2 and ApDMT2 coexpression in intestinal bacteria can synthesize glycinebetaine in a large number, improve the salt tolerance of cell.

The present invention is an acceptor with the plant expression vector pCPE that contains CaMV 35S promoter, MCS and plant anti-herbicide gene EPSPs (5-enolpyruvyl-shikimate-3-phosphate synthase gene); Insert ApGSMT2 and ApDMT2 gene (starting), produce plasmid pCPE-ApGSMT2, pCPE-ApDMT2 and pCPE-ApGSMT2-ApDMT2 by the CaMV 35S promoter.These plasmids are changed over to respectively in the agrobacterium tumefaciens lba4404, transform the tobacco cell that can not synthesize glycinebetaine then, produce transfer-gen plant.In the blade of different transfer-gen plants, measure glycinebetaine content, confirm whether transgenic produces the product function.Genetic transformation and transfer-gen plant analytical procedure are: getting the tobacco aseptic seedling is material, adopts Ye Panfa to transform.(Monsanto screens on inducing culture USA) the transgenic budlet, on root media, takes root containing selective agent 4mg/L glyphosate.Adopt PCR and Southern hybridization analysis method to confirm transfer-gen plant.Detect the expression level of transgenic in tobacco leaf through RT-PCR (ReverseTranscription PCR) method, utilize ¹Glycinebetaine content in the quantitative blade of H NMR assay method.The result shows that the tobacco of coexpression ApGSMT2/ApDMT2 not only has the transgenic transcript than high abundance in the cell, and (strain is that 9 concentration are 0.1832 μ molg to have accumulated the glycinebetaine of higher concentration ^-1FW), be higher than the glycinebetaine content that changes the betA gene plant significantly.And transfer-gen plant and only contain ApGSMT2 or the transfer-gen plant of ApDMT2 gene does not detect glycinebetaine accumulation not.

For detect changeing the influence with resistance of growing of ApGSMT2/ApDMT2 gene pairs tobacco plant, with transfer-gen plant seed and transgenic adjoining tree planting seed not in flowerpot, the variation that observation is grown.Transgenic tobacco plant does not have phenotypic difference under the normal growth condition.Yet; Through detecting survival rate and the growth velocity of transgene tobacco leaf dish under high salt and osmotic stress condition; Draw the tobacco that changes the ApGSMT2/ApDMT2 gene and compare drought-and salt-tolerance with contrast and increase substantially, the change of transgene tobacco drought-and salt-tolerance is because ApGSMT2/ApDMT2 genetic expression and glycinebetaine accumulation cause.

Can influence the reaction of transgenic tobacco plant in order to understand ApGSMT2/ApDMT2 genetic expression to drought stress; Place the MS inorganic salt solution that contains 10% (W/V) PEG 6000 to carry out osmotic stress the transgene tobacco seedling and handle, variations such as the variation of detection blade relative water content and cell membrane damage.Drawing tobacco plant blade relative water content in the osmotic stress process reduces gradually; Cell membrane damage constantly increases the weight of; And the comparison of transfer-gen plant relative water content is low according to the plant changing down; Cell membrane damage is obviously lighter, and growth is very fast, shows that the tobacco plant drought tolerance of ApGSMT2/ApDMT2 genetic expression improves a lot.This gene has good using value in the crop resistance breeding.

Crop genetic transforms and transgenic line obtains

Select for use different transgenic methods to obtain transgenic plant according to transgenic acceptor.

Method for transformation commonly used has: 1) agriculture bacillus mediated genetic transforming method as through inflorescence dip method arabidopsis thaliana transformation, obtains positive transformed plant through dull and stereotyped antibiotic-screening and PCR checking and carries out subsequent analysis.2) direct conversion method promptly extracts recombinant plasmid dna, adopts particle gun blast technique, polyoxyethylene glycol revulsion, electro fusion method, silicon-carbon fibre method etc. directly with the DNA transfered cell.3) planting is conversion method, comprises pollen tube passage method, ovary injection etc.In addition, also has the Transformation Program that the combination of inhomogeneity method is used, as particle gun blast technique and Agrobacterium are used in combination to improve transformation efficiency.The present invention is to be that material carries out genetic transformation with crops such as corn, wheat, jowar, soybean, cottons, and the method for transformation of employing is mainly agrobacterium-mediated transformation and particle gun blast technique.

It is example summary testing sequence and method that the present invention obtains resistant strain system with the agrobacterium-mediated transformation transformed wheat:

Wheat seed soaks 7-10min with 0.1% mercuric chloride solution again with 70% alcohol immersion 1-3min, then with sterilized water washing 5 times.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.The sterilization seed is placed on to fill with sterilized water and soaks in the Cans of filter paper, is placed under the dark condition (24 ± 2 ℃) 1-2 days after sealing.Treat seed sprout grow radicle and plumule after, place it on the M1 substratum in dark condition (24 ± 2 ℃) and continue down to cultivate.When treating that plumule is stretched to 2-5cm, peel off coleoptile and 1-2 sheet spire, expose the point that sprouts with scalper, again with scalper from the middle part vertical cut wound shoot tip meristem, cut primary root.The aseptic seedling of cut wound shoot tip meristem is used for Agrobacterium-mediated Transformation.

To have agrobacterium tumefaciens lba4404 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum of purpose plasmid, concussion speed is 180rpm, makes bacterium be in logarithmic phase.Under 3000rpm centrifugal 5 minutes then, abandon supernatant.(acetosyringone, MS liquid nutrient medium AS) is resuspended with adding the 100mg/l Syringylethanone with thalline again.Then bacterium liquid is poured in the petridish of diameter 15cm, the inclination petridish makes to be immersed in the bacterium liquid by the aseptic seedling stem end of cut wound, vacuumizes (0.05MPa) and infects 7～10min.

Stem apex after the dip-dye blots with aseptic filter paper, seedling is inserted wheat germination substratum (MS substratum inorganic salt composition, N vitamin (Li Shirun etc., 1990), sucrose 30g/L, agar 7g/L, pH 5.8-6.0) upward cultivated altogether 3 days in the dark.Culture temperature is 22-24 ℃.Aseptic seedling after will cultivating altogether after 3 days is transplanted in the flowerpot of upper strata vermiculite lower soil, and covers the plant top with loose moistening vermiculite.Let plant under natural lighting, grow then, warm 22-28 ℃ of day, temperature 15-21 ℃ of night.Water the inorganic salt solution of 1/2MS substratum every other day.

When being transplanted to transformed plant length in the vermiculite to tri-leaf period, the weedicide aqueous solution that evenly sprays suitable concn screens.Sprinkling is fallen drop with plant leaf and is advisable.Spray the back plant and be placed on the greenhouse growth, day temperature 15-20 ℃, about 10 ℃ of nocturnal temperatures about day illumination 12h, watered 1 time in per 3 days.Period of seedling establishment is got leaf DNA and is carried out the PCR detection.

With the epsps gene is the transformed plant sprinkling 0.12%Roundup aqueous solution of selection markers gene, promptly adds 1.2ml " Roundup " (41% glyphosate isopropyl amine salt) in every liter of tap water.Spray back about 15 days not the transgenic adjoining tree begin death, the adjoining tree mortality ratio is about 90% after 25 days.Transformed plant is behind the spraying herbicide aqueous solution, and some individual variations are similar with adjoining tree, and other individualities then continue growth, change not obvious.The survival plant is long is transplanted to the field to the 4-5 leaf during phase.

Transformed plant (T0) selfing of antiweed screening produces T1 for seed, and the latter sprouts in flowerpot, and seedling is long to 3-4 leaf spraying herbicide aqueous solution during the phase, and weedicide concentration and plant strain growth condition are with T0 generation.The survival plant grows to draw materials behind 3 young leaves and carries out Molecular Detection.PCR detects positive plant and partly transplants into flowerpot every strain one basin; Part is transplanted to the land for growing field crops, manages according to the Routine Management method, survives the winter naturally, gets leaf DNA after turning green and carries out Molecular Detection.Individual plant is isolated pollination self and is got T2 for seed.At field seeding, survive the winter naturally by seedling for seed for part T2, gets young leaves after turning green and carry out PCR detection, Southern hybridization and sxemiquantitative RT-PCR checking.The seed of transgenic homozygous lines is used for salt tolerant, drought-enduring physiological is measured.

The resistance of transfer-gen plant detects

The present invention detects and is utilized as the resistance detection of example summary transfer-gen plant and general procedure and the method for utilizing with the salt tolerant of transgenic wheat.

With the non-transgenic plant is contrast, selects to carry out resistance from the transgenic homozygous lines of the independent transformant of difference and measures.In seed germination test, plant in sand basin of the same size after seed dried, pouring contains the aqueous solution of different concns NaCl (0,100mM, 200mM, 300mM) respectively, and each strain is 300 seeds of every concentration kind.Be consistent between each basin of irrigation amount, observe the reguarity of seed sprouting situation and seedling every day, and avoid drenching with rain.The sand that statistics seed final seedling rate after 20 days, coleoptile expose covering is promptly thought and is sprouted.

According to the mensuration of seed bud ratio, select the salt concn during suitable concn NaCl solution is handled as salt stress.Choose respectively from the transgenic homozygous lines and non-transgenic contrast the carrying out salt tolerant physiology of the independent transformant of difference and measure.With each strain is planting seed (diameter 10cm, high 15cm) in sand basin of the same size, 20 of every basin kinds, and each strain system kind of 8 basin waters the Hoagland nutritive medium every day, and plant is long to the final singling during phase of 3 leaves, stays consistent seedling 15 strains of big or small growing way in each basin.Plant is long during the phase, to be divided into two groups with husky basin by each strain system to 5 leaves at random.Wherein one group begins to water the Hoagland nutritive medium that contains NaCl, continuous pouring 10 days, and another group continues pouring Hoagland nutritive medium as the untreated control group.In the salt stress treating processes, measure glycinebetaine content and other physical signs of handling the plant and the plant of being untreated and comprise photosynthesis index, ion content, RWC, chlorophyll content, cytosol gesture, soluble sugar and proline content, cytolemma ion seepage, mda content and living weight.

The wheat strain system that the plantation transgenic is isozygotied in the saltings carries out the salt tolerance analysis, and can the research transgenic improve the mechanism of wheat salt tolerance in the time of infertility and salt tolerance raising.Winter wheat to the critical porion of salt stress be respectively emerge and the seedling stage and early spring period of seedling establishment, the salt tolerance power finally shows the economic yield in the saltings.

Mensuration result shows that the seed of transgenic line germination rate under condition of salt stress is significantly higher than wild-type, and the seedling growth better.It is about 95% that the center plant division ties up to the following germination rate of 200mM NaCl concentration processing, is about 1.5 times of wild-type, and seedling injury is lighter.5 leaf phase seedlings are carried out salt stress handle, NaCl concentration with every day 50mM be incremented to final concentration 200mM, then pouring every day once, salt concn is constant.After salt stress was handled, the transfer-gen plant growing way obviously was superior to adjoining tree, and the over-ground part living weight comparison of handling first quarter moon rear section transfer-gen plant is high more than 30% according to plant, and the root living weight is also compared according to plant high by about 30%.

Measure under the condition of salt stress not that homophyletic is the variation of the physiological parameter of seedling, no matter draw transfer-gen plant or adjoining tree, the Na in blade and the root ⁺Content all obviously raises, K ⁺Content slightly raises earlier and then reduces.Compare transfer-gen plant Na with adjoining tree ⁺Increasing amount is few, K ⁺The reduction amplitude is little, the Na of part strain system ⁺, K ⁺Content is compared equal difference with contrast remarkable, and K ⁺/ Na ⁺Ratio also is significantly higher than adjoining tree.Under condition of salt stress, the chlorophyll content of all strains system all is to have earlier to raise by a small margin to descend with different rates then, but the chlorophyll content of transfer-gen plant is significantly higher than adjoining tree handling the after date phase.Photosynthetic rate, stomatal conductance, intercellular CO ₂The mensuration result of concentration and PS II photochemistry efficient shows, coerces under the processing at long period NaCl, and photosynthetic rate reduces because but stomatal conductance reduction and PS II complex body activity receive cause jointly significantly.But in salt stress was handled, transfer-gen plant showed obviously higher photosynthetic rate, stomatal conductance and maximum optical chemical efficiency (Fv/Fm), promptly in salt stress is handled, can synthesize more glucide.

Under the condition of salt stress, the leaf cell solute potential reduces along with the increase of the time of coercing gradually, but the solute potential of part transgenic line significantly is lower than adjoining tree, promptly has stronger water conservation and the ability that absorbs moisture.Compare with adjoining tree, rotaring gene plant blade contains more soluble sugar and proline(Pro).These results show, under condition of salt stress, have accumulated more soluble substance in the transgenic line cell and (have comprised Na ⁺With proline(Pro) and soluble sugar etc.), make cell keep lower solute potential, help the growth of cell homergy and photosynthesis and plant.Under condition of salt stress; Transfer-gen plant shows lower MDA level and ion percolation ratio; Show that the damaged membrane degree is less than adjoining tree; The higher glycinebetaine level of accumulation is remarkable negative correlation in this and the transfer-gen plant, has higher PS II activity consistent with transfer-gen plant.The stability and the biomacromolecule function that show the excess accumulation cell membrane of glycinebetaine in transfer-gen plant have produced useful provide protection.

Show at the transgenic line of beach saline land plantation and the test-results of check variety; The survival and grow of some transgenic lines in the saltings obviously is superior to check variety; Economic characters such as individual plant tiller number, boot leaf area, single plant yield etc. significantly are superior to check variety, show the salt tolerance that significantly improves.Promptly change the salt tolerance that glycocoll sarkosine N-methyl transferase gene (ApGSMT2) and N-methylsarcosine N-methyl transferase gene (ApDMT2) wheat are significantly increased.

Beneficial effect of the present invention

The invention discloses glycocoll sarkosine N-methyl transferase gene (ApGSMT2) and the coded protein sequence of N-methylsarcosine N-methyl transferase gene (ApDMT2) sequence and supposition in a kind of cyanobacteria glycinebetaine route of synthesis.These two gene sources are in having a liking for salt aphanothece (Aphanothece halophytica); After merging with higher plant constitutive promoter (cauliflower mosaic virus (CAMV) 35SRNA gene promoter, corn Ubiquitin gene promoter); Change over to jointly in the crops such as corn, wheat, cotton, tobacco; Significantly improved degeneration-resistant (drought, salt, cold, high temperature) property of plant, shown that these two genes have good using value in the crop breeding for stress tolerance.

The present invention is the basis with clone's ApGSMT2 and ApDMT2 gene; Needs according to the crop breeding for stress tolerance; Gene order is modified; (1) with codon replacement low codon of frequency of utilization in higher plant of cereal plants and corn preference, ApGSMT2d and ApDMT2d gene have been synthesized; (2) protein is merged respectively at the coding region of chloroplast(id) positioning sequence and ApGSMT2 and ApDMT2 gene, ApGSMT2d and ApDMT2d gene; Produce ApTpGSMT2 and ApTpDMT2 gene, ApTpGSMT2d and ApTpDMT2d gene, realized the location of transgene expression product in chloroplast(id); (3) protein is merged respectively at the coding region of mitochondrial matrix positioning sequence and ApGSMT2 and ApDMT2 gene, ApGSMT2d and ApDMT2d gene; Produce ApTpGSMT2m and ApTpDMT2m gene, ApTpGSMT2dm and ApTpDMT2dm gene, realized that the transgene expression product is in Intramitochondrial location; (4), the location and the high local concentrations accumulation of glycinebetaine under the stress-inducing condition of transgene expression product different zones in cell have been obtained through the application of polygene conversion carrier and the application of transgenic polymerization method.Like this, transgenic breeding for stress tolerance novel material or the new variety that can recombinate out various as required.

Description of drawings

The T-DNA district collection of illustrative plates of Fig. 1, plasmid pCUA-APGSMT2-ApDMT2-epsp

P35S promoter, the cauliflower mosaic virus 35S promoter; PUbi, corn ubiquitin ubiquintin gene promoter; Epsp, the selection markers gene; RB and LB, the left and right border of the T-DNA of Ti-plasmids; Tnos, the no terminator.

Embodiment

The creation of embodiment 1:ApGSMT2/ApDMT2 gene clone and the degeneration-resistant material of corn gene

1) ApGSMT2/ApDMT2 gene clone

Cultivation is had a liking for salt aphanothece (Aphanothece halophytica) to OD600=1.2, gets 30ml bacterium liquid and packs in the 50ml centrifuge tube centrifugal collection thalline into.With STE (0.1MNaCl, 10mMTris-HCl (pH 8.0), 1mMEDTA (pH8.0)) the resuspended thalline of liquid, centrifugal collection repeats 1 time.Add again 20ml 2 * CTAB (20mmol/LEDTApH 8.0 for 100mmol/LTris-HCl pH8.0,1.4mol/LNaCl, 2%CTAB) damping fluid, mixing, incubation 90min in 65 ℃ of water-baths (preheating before add 1% beta-mercaptoethanol), the centre shakes up 2-3 time.Then centrifuge tube is taken out from water-bath, be cooled to and add equal-volume chloroform/primary isoamyl alcohol (24: 1) after the room temperature, put upside down centrifuge tube gently several times, the centrifugal 10min of 10000rpm under the room temperature.Again supernatant is changed in the new 50ml centrifuge tube, with 0.8 times of volume isopropanol precipitating.Deposition changes in 70% ethanol washing 2 times over to, dry then, with 2ml TER (2ml TE+2 μ l RNaseA (10mg/ml)) 37 ℃ of dissolvings.Add 2 times of absolute ethyl alcohols that volume is cold then.Mixing is ticked DNA with the rifle head after leaving standstill a little while gently, behind 70% alcohol flushing 2 times, and vacuum-drying.TE dissolving DNA according to the DNA that carries of institute amount adding 100-500ul.

Design degenerated primer GSMT-F 5 ' ATGGC (T/C/A/G) AA (A/G) AA (A/G) GT (T/C/A/G) GA 3 ' and GSMT-R 5 ' (C/T) TA (G/A) TC (C/T) TT (C/T) TT (C/T/G/A) GC 3 '; DMT-F 5 ' ATGAC (T/C/A/G) AA (A/G) GC (T/C/A/G) GA (T/C) GC 3 ' and DMT-R 5 ' (C/T) TA (T/C/A/G) GG (C/T) TT (G/A) TG (G/A) AA 3 '.With carried DNA is template, uses degenerated primer GSMT-F and GSMT-R respectively, DMT-F and DMT-R, and amplification obtains dna fragmentation.Response procedures is 95 ℃ of preparatory sex change 5min; 95 ℃ of sex change 1min, 45 ℃ of annealing 1min, 72 ℃ are extended 30s, circulate 35 times; 72 ℃ are extended 7min.Adopt Takara to reclaim Kit and reclaim the dna fragmentation that pcr amplification goes out; Select Easy Vector Systems Kit (Promega for use; USA) the PCR fragment is cloned the order-checking of order-checking company.Obtained the nucleotide sequence that length is respectively 798bp and 834bp, each own coding ORF.Based on the GSMT and the DMT sequence of other kinds biology of having reported, name institute clone gene is ApGSMT2, ApDMT2 gene.

2) structure of plant expression vector

Adopt conventional DNA recombinant technology; Plant expression vector pCPE to contain CaMV 35S promoter, MCS and plant anti-herbicide gene EPSPs (5-enolpyruvyl-shikimate-3-phosphate synthase gene) is an acceptor; Insert ApGSMT2 and ApDMT2 gene (starting) in the T-DNA district, produce plasmid pCPE-ApGSMT2-ApDMT2 by the CaMV 35S promoter.Adopt freeze-thaw method to change agrobacterium tumefaciens lba4404 or AGL0 etc. over to this plasmid, be used for corn gene.

3) foundation of transgene receptor system

With key self-mating system used in China's agriculture prodn is material, like Zheng 58, tuck in 478, tuck in 515 etc. self-mating system seed.Isolated culture induces stem apex to produce the sprout tuber that grows thickly, and is that acceptor carries out genetic transformation with the sprout tuber that grows thickly.Used substratum has:

Seed germination substratum: KNO ₃1900mg/l, NH ₄NO ₃1650mg/l, CaCl ₂2H ₂O 440mg/l, MgSO ₄7H ₂O 370mg/l, KH ₂PO ₄H ₂O 170mg/l, FeSO ₄7H ₂O 27.8mg/l, ZnSO ₄7H ₂O 10mg/l, MnSO ₄4H ₂O 22.3mg/l, H ₃BO ₃10mg/l, KI 0.83mg/l, Na ₂MoO ₄2H ₂O 0.5mg/l, CuSO ₄5H ₂O 0.025mg/l, CoCl ₂6H ₂O 0.025mg/l, vitamin 10.0mg/l, pyridoxine hydrochloride 1.0mg/l, nicotinic acid 1.0mg/l, glycocoll 2.0mg/l, inositol 100.0mg/l, vitamin H 0.05mg/l, casein hydrolysate 500mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8-6.0.Be used for seed germination.Liquid nutrient medium then removes agar powder.

The A substratum: the seed germination substratum adds 6-BA 4.5-9.0 μ mol/l and 2, and 4-D 1.0-3.0 μ mol/l is used to induce isolated culture bud point to produce grow thickly the bud tissue block and the bud tissue block succeeding transfer culture of growing thickly.

B substratum: additional 6-BA 4.5 μ mol/l of seed germination substratum and IBA (indolebutyric acid) 1.8 μ mol/l, the bud tissue block that is used to grow thickly differentiation seedling.

Become the seedling substratum: additional 6-BA2.25 μ mol/l of seed germination substratum and IBA 3.6 μ mol/l, the budlet that is used to grow thickly develops into seedling.

Root media: the seed germination substratum adds IBA2.8～3.6 μ mol/l, is used for the little seedling rooting of unrooted.

Minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic and weedicide isoreactivity composition filtration sterilization add behind medium sterilization.

Seed sterilization and sprouting: corn seed with 0.1% mercury chloride immersion 10-15 minute, washs 3-5 time with sterilized water with 70% alcohol immersion 10 minutes more then.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of (30-40 milliliter/250 milliliter triangular flask) sterilized water in the bottle, is placed under the dark condition (23-30 ℃) 1--2 days after sealing.(showing money or valuables one carries unintentionally) back seed of sprouting is placed on the minimum medium and continues to sprout down in dark condition.

Stem tip culture with grow thickly that the bud tissue block is induced, subculture, differentiation: when the plumule of germinated seeds is stretched to 3-5 centimetre; Peel off coleoptile and spire; Cut the epicotyl and the stem apex that are about 5 millimeter; Be inoculated into (24-27 ℃) cultivation under dark on the A substratum, and in time cut the plumular axis of elongation and peel off spire.Cultivate after 6-10 days, stem apex begins the irregular growth of expanding, and several wartys or digitation on the meristematic tissue that expands, occur.After 20 days, begin to form indefinite bud and embryoid on the surface of warty or digitation.General per 4 all succeeding transfer culture once.In succeeding transfer culture, budlet is on the high side 2 if the bud tissue block of growing thickly is grown thickly, and 4-D concentration is got 3.0 μ mol/l; Bud tissue block callusization is heavier if grow thickly, though a large amount of meristematic cell groups is arranged, indefinite bud seldom appears in the surface, can be with 2, and 4-D concentration is reduced to 1.0 μ mol/l, continues to cultivate then to produce a large amount of wartys or digitation again.The bud tissue block of growing thickly on the A substratum, minority has the generation of adventive root.Exist equally with spire, adventive root also influences the generation of expanding growth and embryoid and the budlet that grows thickly of tissue block, need remove early.The bud tissue block of growing thickly was transferred on the B substratum after 2-3 days, and color and luster is flavescence gradually, and quality is more pliable and tougher, and microspike appears in the surface after 5-6 days.ESEM is observed visible each phase embryoid and indefinite bud down.Embryoid and indefinite bud are grown rapidly, form the budlet that grows thickly on the tissue block surface.

4) with the bud tissue block of growing thickly be the conversion and the plant regeneration of acceptor

(every liter of substratum contains: tryptone 10g at additional antibiotic LB substratum will to have the agrobacterium tumefaciens (like AGL0 and LBA4404) of goal gene; Yeast extract 5g; NaCl 10g, pH 7.0, autoclaving) in 28 ℃ down concussion cultivate; Concussion speed be 110rpm (rev/min), make bacterium be in logarithmic phase.Under 3000rpm centrifugal 10 minutes then, abandon supernatant.Thalline is with liquid seeds germination medium (be that the seed germination medium component reduces by half, the remove agar powder) washing of 1/2 concentration, centrifugal again collection.(acetosyringone, As) the liquid inducing clumping bud substratum of 1/2 concentration of 100 μ mol/l suspends, and dilutes 5-10 and doubly is used for transforming with adding Syringylethanone with thalline again.

Getting the bud tissue block of growing thickly of cultivating 13--20 days behind the subculture is transgene receptor.Transform with agrobacterium-mediated transformation, transform the back material and recover darkling to cultivate.Grow thickly budlet or the bud tissue block of growing thickly of agroinfection were cultivated 7-12 days bacteria growing inhibiting in dark on the substratum that is added with cephamycin (Cefotaxime) 250mg/l or Pyocianil (Carb) 500mg/L.Grow thickly budlet or the bud tissue block of growing thickly of recover cultivating after back or the antibacterial cultivation are being added with step sizing 3-4 generation on the substratum of selective agent, obtain transgenic cell and budlet.The exhausted big bud tissue block of growing thickly of counting is dead gradually in screening and culturing.Remove 2 with what the tissue block of survival was transferred to no selective agent, recover to cultivate generation budlet in back on the A substratum of 4-D.

Budlet is placed on into irradiation growth down on the seedling substratum, light intensity 2000-3000lx, illumination 14-15 hour/day.Seedling length changes in the root media during to 3-4 sheet leaf takes root.Cultivated about 15 days, about 40% seedling produces new root.For the seedling of not taking root, its base portion of cut wound is transferred on the new root media and is cultivated, and most of plant produce root system after 10 days.Take root behind the seedling flush away substratum, being transplanted to the vermiculite is to grow in the flowerpot of cultivation medium.Plant grows under natural lighting, day warm 22-28 ℃, at temperature 15-21 ℃ of night, waters the inorganic salt composition of the seed germination substratum of 1/2 concentration every other day.Grow about 2 weeks, nursery transplant produces flourishing root system, is colonizated in the field then.

5) resistance of transfer-gen plant detects and selects and utilizes

The blade of getting the transplant survival plant carries out molecular Biological Detection and confirms transfer-gen plant.With transfer-gen plant bagging self-fertility.Get planting seed in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and glycinebetaine assay, and the bagging selfing is reserved seed for planting according to qualifications.The transgenic homozygous lines is carried out salt tolerant, drought tolerance respectively detect, and numerous kind of bagging selfing, obtain the transgenic self-mating system that resistance significantly improves than transgenic self-mating system not.The latter can be used for preparing the drought-enduring cross-fertilize seed of corn salt tolerant.

6) salt tolerant of transgenic corns, drought tolerance detect and utilize

With the milpa bagging selfing of isozygotying of changeing the ApGSMT2d/ApDMT2d gene, planting seed in the sand basin, the pouring 0.8%NaCl aqueous solution, and be contrast with transgenic self-mating system seed not.The not homophyletic of transfer-gen plant ties up to the salt solution pouring and shows different salt tolerances down; Most genetically modified strains are to show than illumination to show the salt tolerance that improves; The 5 leaf phase surviving rates that strains more than half are are more than 70%; And contrast self-mating system seed is seldom emerged, and also dead gradually after emerging, the surviving rate of 5 leaf phases is less than 10%.Real-time quantitative RT-PCR detects and shows that transgene expression intensity is high, is not having detection signal in the transfer-gen plant.Pick out the excellent transfer-gen plant bagging selfing of salt tolerance and isozygoty, and carry out combining ability test, select combining ability identical with the donor self-mating system or to have the transgenic self-mating system of raising to be used for haloduric corn breeding hybridized.

With the seed of the seed of the milpa of isozygotying that changes the ApGSMT2/ApDMT2 gene and not genetically modified contrast self-mating system broadcast flowerpot in; Carry out drought stress in seedling stage (3-7 leaf phase), male and female fringe growth period (9-13 leaf phase), the pollination phase of blooming respectively and handle, detect arid and handle the difference of influence, damaged membrane degree, single plant yield and other economic characters and the physical signs of corn growth etc.Comprehensive many-sided test result, genetically modified corn comparison shows the drought tolerance of obvious raising according to plant.Select the excellent transfer-gen plant bagging selfing of drought tolerance and isozygoty, and carry out combining ability test, selection combining ability is identical with the donor self-mating system or have the transgenic self-mating system of raising to be used for the seed selection of the corn seed of single cross.

The creation of the synthetic and degeneration-resistant material of corn gene of embodiment 2:ApGSMT2d, ApDMT2d gene

1) structure of the synthetic and expression of plants structure of ApGSMT2d, ApDMT2d gene

Select the preference codon replacement part A pGSMT2 of cereal plants and corn and the codon of ApDMT2 for use, synthetic ApGSMT2d and ApDMT2d gene.Be connected gene fusion construct with the RD29 promotor then, the latter is recombinated among the plant expression vector pCPE, produce pCPE-ApGSMT2d-ApDMT2d.Recombinant plasmid imported be used for genetic transformation in the agrobacterium tumefaciens.

2) acquisition of corn aseptic seedling

Bagging obtains seeds such as the key self-mating system of corn such as Zheng 58, neat 319, prosperous 7-2, with 70% alcohol immersion 10 minutes, again with 0.1% mercury chloride immersion 10-12 minute, washs 3-5 time with sterilized water then.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into small amount of aseptic water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium, under dark condition, continue to sprout.When treating that plumule is stretched to 3-5 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.

3) Agrobacterium is cultivated and activation

(the Mini-Ti plasmid is pCPE-ApGSMT2d-ApDMT2d will to have binary vector; This plasmid has herbicide glyphosate resistant gene epsps) agrobacterium tumefaciens (AGL1 or LBA4404) 28 ℃ of concussions in additional antibiotic YEP substratum cultivate; Concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant.Thalline is with 1/2 improvement MS liquid nutrient medium washing, centrifugal collection.Thalline is suspended with the 1/2MS improvement liquid nutrient medium that adds the 100mg/L Syringylethanone, dilution 5-10 doubly is used for transforming again.

4) the corn aseptic seedling transforms.

A. be poured on bacterium liquid in the petridish of 4.5 cm diameters, the inclination petridish, the aseptic seedling top that will expose the stem apex growing tip is immersed in 2-5 minute (AGL15 minute, LBA44049 minute) in the bacterium liquid.

B. the bud point after contaminating blots with aseptic filter paper, root is inserted in the modified MS medium in dark, cultivated 2-3 days, and culture temperature is 22-24 ℃, aseptic seedling is placed under the scattered light cultivated 2 days then.

C. the aseptic seedling after irradiation being cultivated is transplanted in the flowerpot that is covered with upper strata vermiculite and lower floor's loam, and vermiculite covers the plant top.Let plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.

5) transformed plant screening and field planting

After transformed plant grows 3 leaves, spraying the Glyphosate 62 IPA Salt aqueous solution, is contrast with unconverted plant.The sprinkling amount is not fallen drop with plant and is advisable.Adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 15 days.Plant after the conversion processing, some individual variations are similar with adjoining tree, and other individualities then continue growth, no considerable change.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.

6) evaluation of transfer-gen plant, management and offspring analysis

The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene, and the PCR positive plant is carried out Southern blotting checking and RT-PCR detection.Bagging selfing or sisters handed over solid after transfer-gen plant was bloomed.Planting seed is in the land for growing field crops or the greenhouse, and plant is long gets blade to the 4-6 leaf and extract DNA during the phase, adopt round pcr to detect and whether have foreign gene, the PCR positive plant carried out Southern blotting detects and the RT-PCR detection, therefrom selects transfer-gen plant.Simultaneously transfer-gen plant is carried out the bagging selfing and reserve seed for planting, analyze the individual ratio of transgenic among the next generation, select transgenic and isozygoty and be.

7) resistance of transfer-gen plant is measured

Selecting the transgenic homozygous lines to be used for measuring, is contrast with the non-transgenic self-mating system.Transfer-gen plant seed and contrast sowing seed same period are seeded in respectively in the flowerpot that size is consistent, the soil amount equates, make plant be in suitable growth season.The plant strain growth environment is consistent with management process.Kind grow to 10 leaf after dates in the milpa of flowerpot, every day is watered the water yield (about 600ml) in control, makes that relative water content remains on about 15-16% in the soil; 9-16 on daytime plant blade wilting between period; Night, blade recovered to stretch, and continued to handle for 2 weeks, recovered then normally to water.The all backs plant that recovers normally to water is taken out hero.Again that flowerpot is placed apart, so that the plant self-fertility.Results mature fruit cluster and cauline leaf, living weight and grain yield are measured in the oven dry back.With plant control water the 2nd day (blade began to wilt about at 9 o'clock in the morning) be designated as drought stress and handled the 0th day; Drew materials in the 7th day the 0th, 1,5,10,14 day that handles and the back of recovering normally to water respectively, adopt conventional plant physiology method to measure and degeneration-resistant relevant each item physical signs.And male and female fringe developmental state, plant height, seed weight, pollen vigor etc. are observed.With the 10 leaf phase of part plant stop to water, observe continuously arid to the influence of vine growth and development and survival rate.In entire test, plant is under natural lighting and the temperature and grows, and avoids plant to be drenched with rain during the control water treatment.

Under the normal growth condition, grow and the morphological specificity of transfer-gen plant and adjoining tree do not have significant difference.After drought stress was handled, transfer-gen plant and adjoining tree notable difference all occurred in proterties such as plant height, tassel branch amount, the loose powder of blooming time, the loose powder-pitch time of weaving silk, pollen vigor, fruit ear length, 100-grain weights.Transfer-gen plant tassel branch amount is not few, stamen development is bad, pollen abortion, loose powder-pitch time of weaving silk is long, the individual plant grain yield is low; And transfer-gen plant shows the drought tolerance of obvious raising; Pollen development is normal, the individual plant grain yield apparently higher than the contrast.It is more normal to be that transfer-gen plant grows in arid treating processes, has obvious enhanced drought tolerance.Measure the blade relative water content, the solubility total sugar content, the ion percolation ratio, mda content, physical signs such as proline content, be illustrated in rotaring gene plant blade relative water content comparison under the drought stress according to plant reduce slow, water retention capacity is stronger; Ion percolation ratio and mda content show that the rotaring gene plant blade cell membrane damage is less; Soluble sugar and proline content explain that transfer-gen plant possibly be to keep homergy through accumulating more osmotic protection property material apparently higher than adjoining tree under drought stress.

To the salt solution of corn pouring in seedling stage 0.8%, the salt tolerance of transfer-gen plant is apparently higher than transgenic adjoining tree not.Measure the discoveries such as over-ground part and underground part fresh weight, dry weight under the salt treatment condition of plant in seedling stage, transfer-gen plant grows normally, situation such as growth retardation, plant dwarfing do not occur.

Present embodiment has createed the transgenic corns material that drought-enduring salt tolerance significantly improves.

The structure of embodiment 3, ApTpGSMT2d, ApTpDMTd2 gene and the creation of the degeneration-resistant material of corn gene

1) structure of the synthetic and expression of plants structure of ApTpGSMT2d, ApTpDMTd2 gene

For ApGSMT2 and ApDMT2 are efficiently expressed in maize cell; And the accumulation of realization glycinebetaine in chloroplast(id) (plastid); ApGSMT2d and ApDMT2d gene are merged with peptide (transit peptide) coding region of leading of location chloroplast protein respectively; ApGSMT2 and ApDMT2 albumen are positioned in the chloroplast(id) (plastid) after synthetic, and catalysis is synthetic a large amount of glycinebetaine in chloroplast(id).According to the Arabidopis thaliana RUBISCO small ylidene gene sequences Design PCR primer of having reported; From Arabidopis thaliana, clone the peptide-coding region of leading of RUBISCO small subunit; Be connected respectively to the 5` end of ApGSMT2d gene and ApDMT2d gene opening code-reading frame after the sequence verification; Produce fusion gene, i.e. ApTpGSMT2d and ApTpDMT2d gene.Replace ApGSMT2d and ApDMT2d among the plasmid pCUA-ApGSMT2d-ApDMT2d-epsps respectively with the fusion gene that makes up then, produce plant genetic transferring structure pCUA-ApTpGSMT2d-ApTpDMT2d-epsps.Adopt freeze-thaw method that recombinant plasmid is imported and be used for the maize genetic conversion in the agrobacterium tumefaciens.

2) resistance of generation of corn gene plant and transgenic homozygous lines is measured

With embodiment 2, obtained the transgenic corns material that salt tolerance of drought significantly improves.

The structure of embodiment 4, ApTpGSMT2dm, ApTpDMT2dm gene and the creation of the degeneration-resistant material of corn gene

1) structure of the synthetic and expression of plants structure of ApTpGSMT2dm, ApTpDMTd2m gene

This laboratory work finds that when corn suffered severe drought or salt stress, mitochondrial function reduced significantly, improves the working efficiency of plastosome when cell is coerced, and helps improving the resistance of corn.For ApGSMT2 and ApDMT2 are efficiently expressed in maize cell; And realize that glycinebetaine accumulates in Intramitochondrial compartmentation; ApGSMT2d and ApDMT2d gene are merged with proteic peptide (transit peptide) coding region of leading of LOP plastochondria respectively; Be positioned in the plastosome after making ApGSMT2 and ApDMT2 albumen synthetic, at the tired more glycinebetaine of plastosome inner product.According to reporting sequences Design PCR primer; From the corn spire, clone the peptide-coding sequence of leading of citrate synthase gene; Be connected respectively to 5 ' end of ApGSMT2d gene and ApDMT2d gene opening code-reading frame after the sequence verification; Produce fusion gene, i.e. ApTpGSMT2dm and ApTpDMT2dm gene.Then the fusion gene that makes up is recombinated in the DATEWAY entry vector that has stress induced promoter RD29, start its expression respectively by RD29.Through fixed point reorganization fusion gene is recombinated in the DATEWAY plant expression vector again, produce plasmid pZH-ApTpGSMT2dm-ApTpDMT2dm-epsps.Adopt freeze-thaw method that this plasmid is imported and be used for the maize genetic conversion in the agrobacterium tumefaciens.

2) resistance of generation of corn gene plant and transgenic homozygous lines is measured

With embodiment 2, obtained the transgenic corns material that salt tolerance of drought significantly improves, but the transgenic homozygous lines that material resistance increase rate produces less than embodiment 2 and embodiment 3.

Embodiment 5, ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 import jointly and create corn breeding for stress tolerance novel material

For ApGSMT2 and ApDMT2 are efficiently expressed in maize cell; Realize the not only accumulation in chloroplast(id) (plastid) but also in tenuigenin of glycinebetaine; The probability of avoiding transgene silencing to take place simultaneously; And the accumulation of excessive concentrations causes that plant strain growth is slack-off in the prevention glycinebetaine tenuigenin, therefore, on the one hand ApGSMT2 and ApDMT2 gene is merged with peptide (transit peptide) coding region of leading of location chloroplast protein respectively; Impel synthetic ApGSMT2 and ApDMT2 albumen to be positioned in the chloroplast(id) (plastid), a large amount of glycinebetaines of accumulation in chloroplast(id); On the other hand ApGSMT2d and ApDMT2d gene are merged with stress induced promoter respectively, be implemented in transgene product accumulation in a large number in kytoplasm under the stress conditions.

1) structure of the synthetic and expression of plants structure of ApTpGSMT2, ApTpDMT2 gene

Utilize the peptide-coding region of leading of RUBISCO small ylidene gene in the Arabidopis thaliana cloned; Be connected respectively to ApGSMT2 gene and ApDMT2 gene (without ApGSMT2d and ApDMT2d sequence; The generation of minimizing transgene silencing incident) 5 ' end of opening code-reading frame; Produce fusion gene, i.e. ApTpGSMT2 and ApTpDMT2 gene.

Adopt conventional Protocols in Molecular Biology; ApTpGSMT2d and ApTpDMT2d gene, ApGSMT2d and ApDMT2d are recombinated respectively in the GATEWAY entry vector; Merge with different plant promoter and gene 3 ' tail district respectively; The fusion gene that formation is adapted at expressing in the cereal crop, wherein ApGSMT2d is connected with the promotor RD29 of stress-inducing respectively with the ApDMT2d gene, and ApTpGSMT2 links to each other with constitutive promoter Ubiquitin respectively with ApTpDMT2.Import in the polygene conversion carrier that is the basis with the GATEWAY plant expression vector through the fixed point reorganization then, produce plasmid pZH-ApGSMT2d-ApDMT2d-ApTpGSMT2-ApTpDMT2-epsps.Adopt freeze-thaw method that the polygene conversion carrier is imported in the agrobacterium tumefaciens and be used for the seeding corn and other crops genetic transformation.

2) resistance of generation of corn gene plant and transgenic homozygous lines is measured

With embodiment 2, obtained the transgenic corns material that salt tolerance of drought significantly improves, the latter's resistance significantly is superior to the material of embodiment 2 and embodiment 3 generations.

Embodiment 6, commentaries on classics ApTpGSMT2, ApTpDMT2 gene are created cotton breeding for stress tolerance novel material

1) expression of plants structure and Agrobacterium are cultivated

The structure of plant expression vector and Agrobacterium cultivation and activation are with embodiment 1.Agrobacterium strains is LBA4404.

2) the cotton aseptic seedling obtains

Get the cotton improved seeds or recover the seed of system, slough surperficial fine hair with the vitriol oil, 70% alcohol immersion 1 minute was soaked 10-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into small amount of aseptic water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium that ammonium salt reduces by half and sprout down in dark condition.When treating that plumular axis is stretched to 3-5 centimetre, peel off a slice cotyledon, emergent stem end growing tip.

3) the cotton aseptic seedling transforms

(1) dip-coating of Agrobacterium bacterium liquid is being separated on the stem apex growing tip that digs pricking wound, and covering with agrobacterium liquid wetted cotton balls above that, cultivating darkling then 2-3 days, culture temperature is 24-26 ℃.Again aseptic seedling is placed under the scattered light and cultivated 2 days.

(2) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot of upper strata vermiculite lower floor loam, lets plant under natural lighting, grow, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.

4) transformed plant screening and field planting

After transformed plant grew 3 leaves, spraying herbicide Glyphosate 62 IPA Salt (concentration is different because of the kind) aqueous solution did not fall drop with plant and is advisable.Unconverted adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 12 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and the very strong resistance of other individual performances continues growth.The survival plant is long during the phase, to arrive field with its field planting to 5 leaves.

5) molecular biology identification of antiweed plant

When antiweed plant length arrives the 7-8 leaf, take a sample, mix the back and extract DNA, employing round pcr detection foreign gene from 3-4 sheet leaf.Gather in the crops the seed (selfing) of PCR positive plant, plant the back in the spraying herbicide Glyphosate 62 IPA Salt screening during phase of 3-4 leaf, the survival plant is got blade extraction DNA and carries out the PCR detection, and positive plant carries out the Southernblotting checking and RT-PCR detects.Transfer-gen plant selfing knot peach, the results seed continues plantation.T2 for plant still at 3-4 leaf spraying herbicide Glyphosate 62 IPA Salt during the phase; Statistics survival plant and dead plant ratio; In conjunction with previous generation Southernblotting result, confirm that can transgenic genetic stability, and therefrom select the high transgenic homozygous lines of target gene expression intensity.Further adopt transgene expression intensity and glycinebetaine content in real-time quantitative RT-PCR technology and the different transgenics of glycinebetaine assay technical Analysis then, the selection strain that suits is to carry out the resistance analytical test.

6) resistance of transfer-gen plant is identified and is utilized

Get the seed of the transfer-gen plant that isozygotys, sowing is in flowerpot, and plant 2 leaves carry out subzero treatment during the phase, and through survival rate statistics and physiological index determining, filtering out cold-resistant strain is to be used for producing.

The seed of transgenic line is broadcast in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and salt tolerance detect, and the bagging selfing isozygotys, obtained the salt tolerant kind.

The plant of transgenic line is carried out the drought stress test in seedling stage (5 leaf phase), flower bud phase and flowering period respectively, measure relevant physical signs analysis, confirm the plant drought tolerance.5 leaf phase cotton seedlings are handled in containing the Hogland nutritive medium of 12%PEG-6000; The blade dehydration of wild-type plant is serious after 24 hours; Plant is seriously wilted, and rotaring gene plant blade stretches, plant do not take place to wilt or degree lighter; The blade relative water content of transfer-gen plant is significantly higher than wild-type, and Net Photosynthetic Rate, stomatal conductance and transpiration rate all are significantly higher than wild-type.Osmotic stress has reduced the solute potential of cotton seedling cell; Wherein the solute potential of transfer-gen plant cell reduces more; Physiologic Studies shows, under the drought stress condition, has accumulated more solute in the cell; Strengthened that cell absorbs water and water-holding power, kept normal morphology and cell turgor thereby help plant.Cotton grows to flower bud during the phase in flowerpot, disposablely stop to water after watering sufficient water, carries out drought stress and handles.At flower bud phase drought stress after 8 days; The blade beet alkali content of transfer-gen plant is significantly higher than the wild-type plant; The blade dehydrating speed is obviously compared according to slow; Blade is wilted evening, and blade cell film ion percolation ratio and mda content significantly are lower than the wild-type plant, explain that cell membrane damage and film fat peroxo-degree are all lighter.In addition, the chlorophyll content of transfer-gen plant and superoxide dismutase (SOD) are active also higher in drought stress is handled.The branches of transfer-gen plant contrasts than wild-type and manys 15%-26% behind the drought stress, and the individual plant number of blooming contrasts than wild-type and manys 10%-20%, and individual plant seed cotton yield fall is obviously little to be contrasted than wild-type.Correlation analysis draws, and transgene cotton individual plant seed cotton yield and trimethyl-glycine level are remarkable positive correlation.These results show; The increase of trimethyl-glycine level can improve the drought tolerance of transgene cotton greatly; The raising of transgene cotton drought resistance is not only because the provide protection of trimethyl-glycine cell membrane stability and integrity under the stress conditions, and the accumulation that will give the credit to trimethyl-glycine has improved the osmotic adjustment ability of transfer-gen plant.

Embodiment 7, commentaries on classics ApTpGSMT2d, ApTpDMT2d gene are created wheat breeding for stress tolerance novel material

1) expression of plants structure and Agrobacterium are cultivated

The expression of plants structure is with embodiment 3.Agrobacterium cultivation and activation are with embodiment 1.Agrobacterium strains is LBA4404.

2) the wheat aseptic seedling obtains and transforms

The seed of wheat improved seeds Jimai 20, Jimai 22 etc. is used 70% alcohol immersion 1-3 minute respectively, soaks 8-12 minute with 0.1% mercury chloride, then with sterilized water washing 3-5 time again.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into small amount of aseptic water in the bottle in dark condition (20-25 ℃) 1-2 days down.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium and sprout down in dark condition.When treating that plumule elongation ends 3-4 centimetre, peel off coleoptile and 1-2 sheet spire, expose the point that sprouts with scalper, again with scalper from the middle part vertical cut wound shoot tip meristem.The aseptic seedling of cut wound shoot tip meristem (germinated seeds) is used for Agrobacterium-mediated Transformation.Bacterium liquid is poured in the petridish of 4.5 cm diameters, the inclination petridish is immersed in the bacterium liquid, 0.5 * 10 the aseptic seedling of exposing the stem apex growing tip ⁵The Pa normal atmosphere was handled 6-10 minute down.Bud point after the dip-dye blots with aseptic filter paper, and germinated seeds is placed on the modified MS medium of adding the 200mg/l Stimulina and in dark, cultivated 2-3 days, and culture temperature is 22-24 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.Aseptic seedling after the irradiation cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, and covers the plant top with vermiculite.Let plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.

3) transformed plant screening, field planting and Molecular Detection

After transformed plant grows 3 leaves, the spraying herbicide Glyphosate 62 IPA Salt aqueous solution, concentration is 0.12%Roundup (41% glyphosate isopropyl amine salt), falls drop with plant and is advisable.Unconverted adjoining tree stops growing after back 6 days in sprinkling, and beginning is dead about 15 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.When the plant of waiting to survive length arrives the 7-8 leaf, the field is arrived in its field planting, and promoted to tiller.

The antiweed plant is got blade in the jointing stage of standing up and extracts DNA after vernalization treatment, adopts round pcr to detect transformed plant.In the antiweed plant that obtains, about about 20% is the PCR positive individuals.Latter's self-fertility divides the fringe results.

4) the transfer-gen plant offspring identifies and analyzes

Plant through vernalization stage (the subzero treatment condition is different because of kind) was stood up under the long day, jointing, blossom and bear fruit.The seed that transfer-gen plant produces is put after immersion is sprouted in the refrigerator (0-2 ℃) and was carried out vernalization treatment 30-65 days.Seed after the part vernalization treatment is handled with the 0.12%Roundup aqueous solution, observes the individual ratio of statistics resistance and susceptibility, and the survival seedling is broadcast into flowerpot; Planting seed after another part vernalization treatment carries out drought stress processing and high-salt stress respectively and handles at land for growing field crops and sand basin, screens drought-enduring and/or salt tolerant transfer-gen plant.Extract the leaf DNA of antiweed plant and salt tolerant and drought resisting plant, adopt round pcr to detect foreign gene.Get upper blade extraction DNA behind the PCR positive plant jointing and carry out Southern blotting checking.The transfer-gen plant selfing is set seeds, and following generation will be continued plantation, continues at plant 3-4 leaf spraying herbicide Glyphosate 62 IPA Salt during the phase; The ratio of statistics survival plant and dead plant; In conjunction with previous generation Southernblotting result, confirm that can transgenic genetic stability, and therefrom select transgenic to isozygoty to be.In conjunction with the result of real-time quantitative RT-PCR and the acquisition of glycinebetaine assay, selecting suitable strain is to carry out resistance to measure.Obtain directly to be used for wheat breeding or to get into security semipilot and environmental release test after the transgenic pure lines.

5) the drought resistance analysis of transgenic wheat

Emerge and be wheat to one of tricky time of lack of water seedling stage, this stage drought impact wheat seedling rate and tiller number can make a big impact to output.The seed of transgenic line and adjoining tree is sprouted in the MS inorganic salt solution that contains 0,10%, 15%, 20%, 25% (W/V) PEG-6000 respectively; Statistics germination rate, root length and bud are long; Discovery is under 25% (W/V) PEG-6000 osmotic stress condition; The seed germination comparison of transgenic line is according to early, and the length of bud and root is also genetically modified greater than not respectively, and difference reaches significance level.

Potted plant wheat seedling is carried out continuous drought handle, the living weight of observation plant forms difference and mensuration ground and underground part.Transfer-gen plant in arid is handled growth better, well developed root system, living weight are significantly higher than adjoining tree.The drought tolerance of different transgenic lines has certain difference, and the best transgenic line of drought tolerance is compared photograph plant high 60% and about 40% respectively with the living weight of underground part on the ground when handling 25 days coercing.Under the drought stress condition, higher, the stretching, extension of the blade relative water content of transgenic line, the wilting degree is low, and chlorophyll content, Net Photosynthetic Rate, stomatal conductance and transpiration rate are compared according to significantly high respectively.

Drought stress has reduced the solute potential of wheat seedling cell, has improved cell soluble sugar and proline content.Wherein to reduce amplitude bigger for the solute potential of transfer-gen plant cell, and the accumulation volume of soluble sugar and proline(Pro) is significantly higher than wild-type.Under the drought stress condition, more soluble substance accumulation helps the cell suction and keeps normal turgescence in the transgenic cell.Transgenic line blade cell ion percolation ratio and MDA content all significantly are lower than adjoining tree, explain that the cell membrane damage of transgenic line and film fat peroxo-degree are lighter.Superoxide dismutase of transfer-gen plant (SOD) and katalase (POD) activity also are higher than adjoining tree in drought stress is handled, show that transfer-gen plant has stronger anti-peroxidation ability under the drought stress condition.These results suggest glycinebetaines have the membrane stability of raising and the active effect of stabilized enzyme.The excess accumulation that these results show glycinebetaine has been brought into play vital role in the osmoregulation of transgenic wheat cell with alleviating in the free radical injury, improved the transfer-gen plant drought-resistant ability.

Embodiment 8, cotransformation ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 gene are created soybean breeding for stress tolerance novel material

1) expression of plants structure and Agrobacterium are cultivated

Plant expression vector, Agrobacterium cultivation and activation are with embodiment 5.Agrobacterium strains is LBA4404.

2) the soybean aseptic seedling obtains and Agrobacterium cultivation and activation

Get the seed of improved seeds,, soaked 10-12 minute, then with sterilized water washing 3-5 time with 0.1% mercury chloride with 70% alcohol immersion 2-3 minute.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into small amount of aseptic water (50-60 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 2-3 days after sealing.After treating that seed is sprouted, place it on the improvement B5 medium and sprout down in dark condition.When treating that plumular axis grows to 3-5 centimetre, peel off cotyledon and emergent stem end growing tip.

To have the destination gene expression structure agrobacterium tumefaciens (AGL1 or LBA4404) in additional antibiotic YEP substratum 28 ℃ down concussion cultivate, concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under the 3000r/min centrifugal 10 minutes then, abandon supernatant.Thalline is with 1/2 improvement B5 liquid nutrient medium (promptly improveing B5 medium basal component reduces by half) washing, centrifugal again collection.Thalline is suspended with the 1/2 improvement B5 liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-10 doubly is used for transforming again.

3) the soybean aseptic seedling transforms

(1) bacterium liquid is poured in the petridish of 4.5 cm diameters, the inclination petridish makes the aseptic seedling top of exposing the stem apex growing tip be immersed in the bacterium liquid 3-6 minute.

(2) the bud point after the dip-dye blots with aseptic filter paper, inserts the seedling butt on the improvement B5 medium and in dark, cultivates 3-4 days, and culture temperature is 20-23 ℃.

(3) aseptic seedling is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, the top covers vermiculite, and the long day (16 hours/day) is growth down, warm 23-28 ℃ of day, at temperature 20-25 ℃ of night, waters 1/2 improvement B5 medium inorganic salt every other day.

4) transformed plant screening and field planting

After transformed plant grows 3 leaves, spray the 0.12%Roundup aqueous solution and handle, do not fall drop with plant and be advisable, unconverted adjoining tree stopped growing in sprinkling in back 6 days, and beginning is dead about 15 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.

The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene.The PCR positive plant keeps solid.

5) transfer-gen plant offspring's molecular biology identification

The seed that transfer-gen plant produces is sowed in the land for growing field crops or the greenhouse.Plant 3-4 leaf is got blade extraction DNA and is carried out PCR detection transgenic ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 during the phase.Get PCR positive plant blade and carry out Southern blotting checking, transfer-gen plant self-fertility.Second filial seedling (5 leaf phase) is added up survival plant and dead plant ratio after spraying the 0.12%Roundup aqueous solution, the survival plant is carried out PCR again detect, and confirms the segregation ratio of foreign gene in progeny plant.The transgenic homozygous lines is carried out real-time quantitative RT-PCR detect and the glycinebetaine assay, select the high strain system of transgene expression intensity to be used to detect resistance.

6) transfer-gen plant offspring's resistance is measured

Physiology method and thremmatology method by routine are carried out.

Embodiment 9, realize that through transgenic polymeric method glycinebetaine creates the high resistance to cold and diseases material in the distribution of cell different zones

1) the transgenic polymerization produces corn high resistance to cold and diseases self-mating system

To produce plant, the plant that has ApGSMT2d/ApDMT2d and ApTpGSMT2dm/ApTpDMT2dm gene that has ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2 gene, the plant that has ApTpGSMT2d/ApTpDMTd2 and ApTpGSMT2dm/ApTpDMT2dm gene respectively from the milpa hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same genotype (self-mating system).Then transgenic polymerization plant is carried out continuous selfing, selection, the acquisition transgenic is isozygotied and is.Also can different transfer-gen plants and transgenic polymerization plant be hybridized once more; Obtain to carry different genetically modified self-mating systems through selfing with selection; As carry the corn inbred line of ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene, realize that different critical functions district in the cell all accumulates the target of the glycinebetaine of higher level under the environment stress condition.The hybridization of transgenic corns, selfing and system of selection be with the corn conventional breeding, but need carry out in the transgenic corns isolated area.Whether and in the self-generation of transgenic polymerization plant, need to adopt PCR method to detect all target genes and exist, only selecting and remain, those have the individuality of whole target genes.Coding region corresponding to gene of the PCR primer of each target gene, one corresponding to promoter region, has good specificity.The resistance measuring method of transgenic polymeric materials is with instance 2.

2) the degeneration-resistant novel material of transgenic polymerization wheat produces

To produce plant, transgenic ApTpGSMT2d/ApTpDMTd2 and the ApTpGSMT2dm of the plant, transgenic ApGSMT2d/ApDMT2d and the ApTpGSMT2dm/ApTpDMT2dm that carry transgenic ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2, the plant of ApTpDMT2dm respectively from the wheat plant hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same kind.Then transgenic polymerization plant is carried out continuous selfing, selection, the acquisition transgenic is isozygotied and is.Also can different transfer-gen plants and transgenic polymerization plant be hybridized once more, selfing and selection, obtain to carry the transgenic polymerized strain of different target genes.The hybridization of transgenic wheat, selfing and system of selection be with the wheat conventional breeding, but need carry out in the transgenic wheat isolated area.And adopt PCR method to detect whether target gene all exists among the transgenic polymerization plant self progeny, those individual selfings that have whole target genes of only selecting and remain are isozygotied.The resistance measuring method of transgenic polymeric materials is with embodiment 7.

3) the transgenic polymerization produces the degeneration-resistant novel material of cotton

To produce the plant of plant, transgenic ApTpGSMT2d/ApTpDMTd2 and the ApTpGSMT2dm/ApTpDMT2dm of the plant, transgenic ApGSMT2d/ApDMT2d and the ApTpGSMT2dm/ApTpDMT2dm that carry transgenic ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2 respectively from the cotton plants hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same kind or recovery system.Then transgenic polymerization plant is carried out continuous selfing, selection, the acquisition transgenic is isozygotied and is.Also can different transfer-gen plants and transgenic polymerization plant be hybridized once more, selfing and selection, obtain to carry the transgenic polymerized strain of different target genes.The hybridization of transgene cotton, selfing and system of selection are with the cotton conventional breeding; But need carry out in the transgene cotton isolated area; And adopt PCR method to detect whether target gene exists among the transgenic polymerization plant self progeny, only selecting and remain, those have individual selfing, the selection of whole target genes.The resistance measuring method of transgenic polymeric materials is with instance 6.

4) the degeneration-resistant novel material of transgenic polymerization soybean produces

To produce the plant of plant, transgenic ApTpGSMT2d/ApTpDMTd2 and the ApTpGSMT2dm/ApTpDMT2dm of the plant, transgenic ApGSMT2d/ApDMT2d and the ApTpGSMT2dm/ApTpDMT2dm that carry transgenic ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2 respectively from the soybean plant strain hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same kind.Then transgenic polymerization plant is carried out continuous selfing, selection, the acquisition transgenic is isozygotied and is.Also can different transfer-gen plants and transgenic polymerization plant be hybridized once more, selfing and selection, obtain to carry the transgenic polymerized strain of different target genes.The hybridization of genetically engineered soybean, selfing and system of selection are with the soybean conventional breeding; But need carry out in the genetically engineered soybean isolated area; And adopt PCR method to detect whether target gene exists among the transgenic polymerization plant self progeny, only selecting and remain, those have individual selfing, the selection of whole target genes.The resistance measuring method of transgenic polymeric materials is seen conventional breeding for stress tolerance method.

Embodiment 10, utilization are changeed ApGSMT2 and are prepared degeneration-resistant cross-fertilize seed with the different corn inbred lines of ApDMT2 and derivation gene thereof

To have the excellent heterotic different corn inbred line hybridization that change ApGSMT2d/ApDMT2d or ApTpGSMT2d/ApTpDMTd2 or ApTpGSMT2dm/ApTpDMT2dm gene respectively over to, and produce and carry this genetically modified cenospecies of father and mother respectively.The latter can directly be used for producing, and promptly is the degeneration-resistant cross-fertilize seed of transgenic that has competitive edge on producing.The self-mating system that also can change ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 gene is male parent or the female parent of cross-fertilize seed; Another self-mating system to change the ApTpGSMT2dm/ApTpDMT2dm gene is female parent or the male parent of cross-fertilize seed, thereby realizes that different critical functions district in the hybrid cell all can accumulate the target of higher concentration glycinebetaine under the environment stress condition.The hybrid of transgenic corns produces with the corn conventional breeding, but need carry out in the transgenic corns isolated area.The resistance measuring method of transgenic cross-fertilize seed is with reference to instance 2.

Sequence table

< 110>Shandong University

< 120>methyl transferase gene and modification and the utilization in a kind of trimethyl-glycine route of synthesis

<141>2009-09-01

<160>18

<210>1

<211>17

<212>DNA

<213>GSMT-F

<400>1

atggcnaara argtnga 17

<210>2

<211>15

<212>DNA

<213>GSMT-R

<400>2

ytartcytty ttngc 15

<210>3

<211>17

<212>DNA

<213>DMT-F

<400>3

atgacnaarg cngaygc 17

<210>4

<211>15

<212>DNA

<213>DMT-R

<400>4

ytanggyttr tgraa 15

<210>5

<211>798

<212>DNA

< 213>have a liking for salt aphanothece glycocoll sarkosine N-methyl transferase gene (ApGSMT2)

<400>5

atggctatca aagaaaaaca agttcaagac tacggcgaga atcccattga agtccgggat 60

agtgatcatt atcaaaacga atacattgaa gggttcgtag aaaaatggga cgagttaatc 120

aattggcaag cgcgatcgac cagtgaaggg gaatttttca tcaaaaccct aaaagaacac 180

aatgccgatc gcgttttaga tgctgccacg ggaacgggtt tccactccat tcgcttaatt 240

caagctgggt ttaatgttgc cagtgttgac ggtagcgttg agatgttaat aaaagcattt 300

gaaaacgcca ctcgcaagga tcaaatttta agtacggttc acgcggactg gcgctggtta 360

actcgtgacg tacaggaacg atttgacgca gtgatttgtt taggaaactc cttcactcac 420

ctgttcccag aagaagatcg ccgtaaaaca ctcgccgaat tttattccgt cttgaaacat 480

gacggaattt taattttaga ccaacggaat tacgacttaa ttctggatga agggttcaaa 540

agcaagcata cttactacta ctgtggcgaa aacgtaaaag cagaaccaga atatgtggat 600

gatggtttag ctcgtttccg ttatcagttc cctgatgaat cggtgtatca cctcaatatg 660

ttccccttgc gtaaagatta tgtgcgccgt cttctccatg aggtaggttt ccaagatgtg 720

accacttatg gtgacttcca agaaacttat catcaagatg atccagactt ctacattcac 780

gttgcgaaaa aagattaa 798

<210>6

<211>265

<212>PRT

<213>ApGSMT2

<400>6

Met Ala Ile Lys Glu Lys Gln Val Gln Asp Tyr Gly Glu Asn Pro Ile

1 5 10 15

Glu Val Arg Asp Ser Asp His Tyr Gln Asn Glu Tyr Ile Glu Gly Phe

20 25 30

Val Glu Lys Trp Asp Glu Leu Ile Asn Trp Gln Ala Arg Ser Thr Ser

35 40 45

Glu Gly Glu Phe Phe Ile Lys Thr Leu Lys Glu His Asn Ala Asp Arg

50 55 60

Val Leu Asp Ala Ala Thr Gly Thr Gly Phe His Ser Ile Arg Leu Ile

65 70 75 80

Gln Ala Gly Phe Asn Val Ala Ser Val Asp Gly Ser Val Glu Met Leu

85 90 95

Ile Lys Ala Phe Glu Asn Ala Thr Arg Lys Asp Gln Ile Leu Ser Thr

100 105 110

Val His Ala Asp Trp Arg Trp Leu Thr Arg Asp Val Gln Glu Arg Phe

115 120 125

Asp Ala Val Ile Cys Leu Gly Asn Ser Phe Thr His Leu Phe Pro Glu

130 135 140

Glu Asp Arg Arg Lys Thr Leu Ala Glu Phe Tyr Ser Val Leu Lys His

145 150 155 160

Asp Gly Ile Leu Ile Leu Asp Gln Arg Asn Tyr Asp Leu Ile Leu Asp

165 170 175

Glu Gly Phe Lys Ser Lys His Thr Tyr Tyr Tyr Cys Gly Glu Asn Val

180 185 190

Lys Ala Glu Pro Glu Tyr Val Asp Asp Gly Leu Ala Arg Phe Arg Tyr

195 200 205

Gln Phe Pro Asp Glu Ser Val Tyr His Leu Asn Met Phe Pro Leu Arg

210 215 220

Lys Asp Tyr Val Arg Arg Leu Leu His Glu Val Gly Phe Gln Asp Val

225 230 235 240

Thr Thr Tyr Gly Asp Phe Gln Glu Thr Tyr His Gln Asp Asp Pro Asp

245 250 255

Phe Tyr Ile His Val Ala Lys Lys Asp

260 265

<210>7

<211>834

<212>DNA

< 213>have a liking for salt aphanothece N-methylsarcosine N-methyl transferase gene (ApDMT2)

<400>7

atgactaaag cagacgcagt ggcgcaacag gcccaagact attacgacag tggtagtgcc 60

gatggctttt attatcgcat ttggggagga gaagacctcc acatcgggat ttatgacact 120

cctgatgaac ccatctatga tgcgagtgtg cgtaccgttg ctcgaatttg cgacaaaatc 180

agtaactggg cgccaggaac aaaagtgtta gatattggag caggttatgg tggctccgct 240

cgttatatgg caaagcatca tggctttgtg gtagattgtt tgaatattag tttggtacaa 300

aacgagcgca atcgccagat gaaccaagaa caggggcttg cggataaaat tcgcgtcttt 360

gacggcagtt ttgaagacct cccctttgag gaaaatagtt atgatgtggt gtggtcacaa 420

gatgcgattc tccacagtgg aaaccgtcgc aaggtgttag aagaggtcga tcgcactctg 480

aaatctggga gcgattttgt ctttactgat cccatgcaaa cggataattg tccagaagga 540

gtgttagagc cagttttagc tcggattcat ctggatagtt taggctcagt gagtttctat 600

cgtcaggtgg gagaagaact cggctggcag tttgtggaat ttgatgagca aactcaccat 660

ttagttaacc actacagtag agtgttacag gagttagaag ctcattatga gcaattacaa 720

cctgaatgtt ctaaggagta tttagatcgc atgaaagtgg ggttaaacca ctggatcaat 780

gcgggtaaga gtggttacat ggcttggggg attctcaagt ttcataagcc ctag 834

<210>8

<211>277

<212>PRT

<213>ApDMT2

<400>8

Met Thr Lys Ala Asp Ala Val Ala Gln Gln Ala Gln Asp Tyr Tyr Asp

1 5 10 15

Ser Gly Ser Ala Asp Gly Phe Tyr Tyr Arg Ile Trp Gly Gly Glu Asp

20 25 30

Leu His Ile Gly Ile Tyr Asp Thr Pro Asp Glu Pro Ile Tyr Asp Ala

35 40 45

Ser Val Arg Thr Val Ala Arg Ile Cys Asp Lys Ile Ser Asn Trp Ala

50 55 60

Pro Gly Thr Lys Val Leu Asp Ile Gly Ala Gly Tyr Gly Gly Ser Ala

65 70 75 80

Arg Tyr Met Ala Lys His His Gly Phe Val Val Asp Cys Leu Asn Ile

85 90 95

Ser Leu Val Gln Asn Glu Arg Asn Arg Gln Met Asn Gln Glu Gln Gly

100 105 110

Leu Ala Asp Lys Ile Arg Val Phe Asp Gly Ser Phe Glu Asp Leu Pro

115 120 125

Phe Glu Glu Asn Ser Tyr Asp Val Val Trp Ser Gln Asp Ala Ile Leu

130 135 140

His Ser Gly Asn Arg Arg Lys Val Leu Glu Glu Val Asp Arg Thr Leu

145 150 155 160

Lys Ser Gly Ser Asp Phe Val Phe Thr Asp Pro Met Gln Thr Asp Asn

165 170 175

Cys Pro Glu Gly Val Leu Glu Pro Val Leu Ala Arg Ile His Leu Asp

180 185 190

Ser Leu Gly Ser Val Ser Phe Tyr Arg Gln Val Gly Glu Glu Leu Gly

195 200 205

Trp Gln Phe Val Glu Phe Asp Glu Gln Thr His His Leu Val Asn His

210 215 220

Tyr Ser Arg Val Leu Gln Glu Leu Glu Ala His Tyr Glu Gln Leu Gln

225 230 235 240

Pro Glu Cys Ser Lys Glu Tyr Leu Asp Arg Met Lys Val Gly Leu Asn

245 250 255

His Trp Ile Asn Ala Gly Lys Ser Gly Tyr Met Ala Trp Gly Ile Leu

260 265 270

Lys Phe His Lys Pro

275

<210>9

<211>798

<212>DNA

<213>ApGSMT2d

<400>9

atggccatca aggagaagca ggtgcaggac tacggcgaga acccaatcga ggtgagggac 60

tccgaccact accagaacga gtacatcgag ggcttcgtgg agaagtggga cgagctgatc 120

aactggcagg ccaggtccac ctccgagggc gagttcttca tcaagaccct gaaggagcat 180

aatgctgata gggttctgga tgctgctact ggcactggct tccattctat taggcttatc 240

caagccggct tcaatgtggc ctctgtggat ggctccgtgg agatgctgat caaggccttc 300

gagaacgcca ccaggaagga ccagatcctg tccaccgtgc acgccgactg gaggtggctg 360

accagggacg tgcaggagag gttcgacgcc gtgatctgcc tgggcaactc cttcacccac 420

ctgttcccag aggaggacag gaggaagacc ctggccgagt tctactccgt gctgaagcac 480

gacggcatcc tgatcctgga ccagaggaac tacgacctga tcctggacga gggcttcaag 540

tccaagcaca cctactacta ctgcggcgag aacgtgaagg ccgagccaga gtacgtggac 600

gacggcctgg ccaggttcag gtaccagttc ccagacgagt ccgtgtacca cctgaacatg 660

ttcccactga ggaaggacta cgtgaggagg ctgctgcacg aggtgggctt ccaggacgtg 720

accacctacg gcgacttcca ggagacctac caccaggacg acccagactt ctacatccac 780

gtggccaaga aggactaa 798

<210>10

<211>834

<212>DNA

<213>ApDMT2d

<400>10

atgactaagg ctgacgctgt ggctcagcag gcccaggact actacgactc cggctccgcc 60

gacggcttct actacaggat ctggggcggc gaggacctgc acatcggcat ctacgacacc 120

ccggacgagc cgatctacga cgcctccgtg aggaccgtgg ccaggatctg cgacaagatc 180

tccaactggg ccccgggcac caaggtgctg gacatcggcg ccggctacgg cggctccgcc 240

aggtacatgg ccaagcacca cggcttcgtg gtggactgcc tgaacatctc cctggtgcag 300

aacgagagga acaggcagat gaaccaggag cagggcctgg ccgacaagat cagggtgttc 360

gacggctcct tcgaggacct gccgttcgag gagaactcct acgacgtggt gtggtcccag 420

gacgccatcc tgcactccgg caacaggagg aaggtgctgg aggaggtgga caggaccctg 480

aagtccggct ccgacttcgt gttcacagat ccaatgcaga cagataattg cccagagggc 540

gtgcttgagc cagtgctggc taggatccat cttgattctc tgggctctgt gtctttctac 600

aggcaggtgg gcgaggagct gggctggcag ttcgtggagt tcgacgagca gacccaccac 660

ctggtgaacc actactccag ggtgctgcag gagctggagg cccactacga gcagctgcag 720

ccggagtgct ccaaggagta cctggacagg atgaaggtgg gcctgaacca ctggatcaac 780

gccggcaagt ccggctacat ggcctggggc atcctgaagt tccacaagcc atag 834

<210>11

<211>294

<212>DNA

< 213>the RUBISCO small ylidene gene of Arabidopis thaliana leads peptide-coding region

<400>11

atggcttcct ctatgctctc ttccgctact atatggcttc ctctatgctc tcttccgcta 60

ctatggttgc ctctccggct caggccacta tggtcgctcc tttcaacgga cttaagtcct 120

ccgctgcctt cccagccacc cgcaaggcta acaacgacat tacttccatc acaagcaacg 180

gcggaagagt taactgcatg caggtgtggc ctccgattgg aaagaagaag tttgagactc 240

tctcttacct tcctgacctt accgattccg gtggtcgcgt caactgcatg cagg 294

<210>12

<211>87

<212>PRT

< 213>the RUBISCO small ylidene gene of Arabidopis thaliana leads peptide

<400>12

Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala

1 5 10 15

Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser

35 40 45

Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile Gly Lys

50 55 60

Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly

65 70 75 80

Gly Arg Val Asn Cys Met Gln

85

<210>13

<211>352

<212>PRT

< 213>fusion rotein ApTpGSMT2 and ApTpGSMT2d

<400>13

Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala

1 5 10 15

Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser

35 40 45

Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile Gly Lys

50 55 60

Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly

65 70 75 80

Gly Arg Val Asn Cys Met Gln Met Ala Ile Lys Glu Lys Gln Val Gln

85 90 95

Asp Tyr Gly Glu Asn Pro Ile Glu Val Arg Asp Ser Asp His Tyr Gln

100 105 110

Asn Glu Tyr Ile Glu Gly Phe Val Glu Lys Trp Asp Glu Leu Ile Asn

115 120 125

Trp Gln Ala Arg Ser Thr Ser Glu Gly Glu Phe Phe Ile Lys Thr Leu

130 135 140

Lys Glu His Asn Ala Asp Arg Val Leu Asp Ala Ala Thr Gly Thr Gly

145 150 155 160

Phe His Ser Ile Arg Leu Ile Gln Ala Gly Phe Asn Val Ala Ser Val

165 170 175

Asp Gly Ser Val Glu Met Leu Ile Lys Ala Phe Glu Asn Ala Thr Arg

180 185 190

Lys Asp Gln Ile Leu Ser Thr Val His Ala Asp Trp Arg Trp Leu Thr

195 200 205

Arg Asp Val Gln Glu Arg Phe Asp Ala Val Ile Cys Leu Gly Asn Ser

210 215 220

Phe Thr His Leu Phe Pro Glu Glu Asp Arg Arg Lys Thr Leu Ala Glu

225 230 235 240

Phe Tyr Ser Val Leu Lys His Asp Gly Ile Leu Ile Leu Asp Gln Arg

245 250 255

Asn Tyr Asp Leu Ile Leu Asp Glu Gly Phe Lys Ser Lys His Thr Tyr

260 265 270

Tyr Tyr Cys Gly Glu Asn Val Lys Ala Glu Pro Glu Tyr Val Asp Asp

275 280 285

Gly Leu Ala Arg Phe Arg Tyr Gln Phe Pro Asp Glu Ser Val Tyr His

290 295 300

Leu Asn Met Phe Pro Leu Arg Lys Asp Tyr Val Arg Arg Leu Leu His

305 310 315 320

Glu Val Gly Phe Gln Asp Val Thr Thr Tyr Gly Asp Phe Gln Glu Thr

325 330 335

Tyr His Gln Asp Asp Pro Asp Phe Tyr Ile His Val Ala Lys Lys Asp

340 345 350

<210>14

<211>364

<212>PRT

< 213>fusion rotein ApTpDMT2 and ApTpDMT2d

<400>14

Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala

1 5 10 15

Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser

35 40 45

Asn Gly Gly Arg Val Asn Cys Met Gln Val Trp Pro Pro Ile Gly Lys

50 55 60

Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Asp Leu Thr Asp Ser Gly

65 70 75 80

Gly Arg Val Asn Cys Met Gln Met Thr Lys Ala Asp Ala Val Ala Gln

85 90 95

Gln Ala Gln Asp Tyr Tyr Asp Ser Gly Ser Ala Asp Gly Phe Tyr Tyr

100 105 110

Arg Ile Trp Gly Gly Glu Asp Leu His Ile Gly Ile Tyr Asp Thr Pro

115 120 125

Asp Glu Pro Ile Tyr Asp Ala Ser Val Arg Thr Val Ala Arg Ile Cys

130 135 140

Asp Lys Ile Ser Asn Trp Ala Pro Gly Thr Lys Val Leu Asp Ile Gly

145 150 155 160

Ala Gly Tyr Gly Gly Ser Ala Arg Tyr Met Ala Lys His His Gly Phe

165 170 175

Val Val Asp Cys Leu Asn Ile Ser Leu Val Gln Asn Glu Arg Asn Arg

180 185 190

Gln Met Asn Gln Glu Gln Gly Leu Ala Asp Lys Ile Arg Val Phe Asp

195 200 205

Gly Ser Phe Glu Asp Leu Pro Phe Glu Glu Asn Ser Tyr Asp Val Val

210 215 220

Trp Ser Gln Asp Ala Ile Leu His Ser Gly Asn Arg Arg Lys Val Leu

225 230 235 240

Glu Glu Val Asp Arg Thr Leu Lys Ser Gly Ser Asp Phe Val Phe Thr

245 250 255

Asp Pro Met Gln Thr Asp Asn Cys Pro Glu Gly Val Leu Glu Pro Val

260 265 270

Leu Ala Arg Ile His Leu Asp Ser Leu Gly Ser Val Ser Phe Tyr Arg

275 280 285

Gln Val Gly Glu Glu Leu Gly Trp Gln Phe Val Glu Phe Asp Glu Gln

290 295 300

Thr His His Leu Val Asn His Tyr Ser Arg Val Leu Gln Glu Leu Glu

305 310 315 320

Ala His Tyr Glu Gln Leu Gln Pro Glu Cys Ser Lys Glu Tyr Leu Asp

325 330 335

Arg Met Lys Val Gly Leu Asn His Trp Ile Asn Ala Gly Lys Ser Gly

340 345 350

Tyr Met Ala Trp Gly Ile Leu Lys Phe His Lys Pro

355 360

<210>15

<211>77

<212>PRT

< 213>fusion rotein ApTpDMT2 and ApTpDMT2d

<400>15

Met Ala Asp Gln Ala Asn Gln Pro Thr Val Leu His Lys Leu Gly Gly

1 5 10 15

Gln Phe His Leu Arg Ser Ile Ile Ser Glu Gly Val Arg Ala Arg Asn

20 25 30

Ile Cys Pro Ser Val Ser Ser Tyr Glu Arg Arg Phe Ala Thr Arg Asn

35 40 45

Tyr Met Thr Gln Ser Leu Trp Gly Pro Ser Met Ser Val Ser Gly Gly

50 55 60

Ile Asn Val Pro Val Met Gln Thr Pro Leu Cys Ala Asn

65 70 75

<210>16

<211>77

<212>PRT

< 213>fusion rotein ApTpDMT2 and ApTpDMT2d

<400>16

Met Ala Asp Gln Ala Asn Ala Pro Thr Val Leu His Lys Leu Gly Gly

1 5 10 15

Gln Phe His Leu Ser Ser Ser Phe Ser Glu Gly Val Arg Ala Arg Asn

20 25 30

Ile Cys Pro Ser Phe Ser Pro Tyr Glu Arg Arg Phe Ala Thr Arg Asn

35 40 45

Tyr Met Thr Gln Ser Leu Trp Gly Pro Ser Met Ser Val Ser Gly Gly

50 55 60

Ile Asn Val Pro Val Met Pro Thr Pro Leu Phe Ala Asn

65 70 75

<210>17

<211>341

<212>PRT

< 213>fusion rotein ApTpGSMT2m

<400>17

Met Ala Asp Gln Ala Asn Gln Pro Thr Val Leu His Lys Leu Gly Gly

1 5 10 15

Gln Phe His Leu Arg Ser Ile Ile Ser Glu Gly Val Arg Ala Arg Asn

20 25 30

Ile Cys Pro Ser Val Ser Ser Tyr Glu Arg Arg Phe Ala Thr Arg Asn

35 40 45

Tyr Met Thr Gln Ser Leu Trp Gly Pro Ser Met Ser Val Ser Gly Gly

50 55 60

Ile Asn Val Pro Val Met Gln Thr Pro Leu Cys Ala Asn Met Ala Ile

65 70 75 80

Lys Glu Lys Gln Val Gln Asp Tyr Gly Glu Asn Pro Ile Glu Val Arg

85 90 95

Asp Ser Asp His Tyr Gln Asn Glu Tyr Ile Glu Gly Phe Val Glu Lys

100 105 110

Trp Asp Glu Leu Ile Asn Trp Gln Ala Arg Ser Thr Ser Glu Gly Glu

115 120 125

Phe Phe Ile Lys Thr Leu Lys Glu His Asn Ala Asp Arg Val Leu Asp

130 135 140

Ala Ala Thr Gly Thr Gly Phe His Ser Ile Arg Leu Ile Gln Ala Gly

145 150 155 160

Phe Asn Val Ala Ser Val Asp Gly Ser Val Glu Met Leu Ile Lys Ala

165 170 175

Phe Glu Asn Ala Thr Arg Lys Asp Gln Ile Leu Ser Thr Val His Ala

180 185 190

Asp Trp Arg Trp Leu Thr Arg Asp Val Gln Glu Arg Phe Asp Ala Val

195 200 205

Ile Cys Leu Gly Asn Ser Phe Thr His Leu Phe Pro Glu Glu Asp Arg

210 215 220

Arg Lys Thr Leu Ala Glu Phe Tyr Ser Val Leu Lys His Asp Gly Ile

225 230 235 240

Leu Ile Leu Asp Gln Arg Asn Tyr Asp Leu Ile Leu Asp Glu Gly Phe

245 250 255

Lys Ser Lys His Thr Tyr Tyr Tyr Cys Gly Glu Asn Val Lys Ala Glu

260 265 270

Pro Glu Tyr Val Asp Asp Gly Leu Ala Arg Phe Arg Tyr Gln Phe Pro

275 280 285

Asp Glu Ser Val Tyr His Leu Asn Met Phe Pro Leu Arg Lys Asp Tyr

290 295 300

Val Arg Arg Leu Leu His Glu Val GlyPhe Gln Asp Val Thr Thr Tyr

305 310 315 320

Gly Asp Phe Gln Glu Thr Tyr His Gln Asp Asp Pro Asp Phe Tyr Ile

325 330 335

His Val Ala Lys Lys

340

<210>18

<211>354

<212>PRT

< 213>fusion rotein ApTpGSMT2m

<400>18

Met Ala Asp Gln Ala Asn Ala Pro Thr Val Leu His Lys Leu Gly Gly

1 5 10 15

Gln Phe His Leu Ser Ser Ser Phe Ser Glu Gly Val Arg Ala Arg Asn

20 25 30

Ile Cys Pro Ser Phe Ser Pro Tyr Glu Arg Arg Phe Ala Thr Arg Asn

35 40 45

Tyr Met Thr Gln Ser Leu Trp Gly Pro Ser Met Ser Val Ser Gly Gly

50 55 60

Ile Asn Val Pro Val Met Pro Thr Pro Leu Phe Ala Asn Met Thr Lys

65 70 75 80

Ala Asp Ala Val Ala Gln Gln Ala Gln Asp Tyr Tyr Asp Ser Gly Ser

85 90 95

Ala Asp Gly Phe Tyr Tyr Arg Ile Trp Gly Gly Glu Asp Leu His Ile

100 105 110

Gly Ile Tyr Asp Thr Pro Asp Glu Pro Ile Tyr Asp Ala Ser Val Arg

115 120 125

Thr Val Ala Arg Ile Cys Asp Lys Ile Ser Asn Trp Ala Pro Gly Thr

130 135 140

Lys Val Leu Asp Ile Gly Ala Gly Tyr Gly Gly Ser Ala Arg Tyr Met

145 150 155 160

Ala Lys His His Gly Phe Val Val Asp Cys Leu Asn Ile Ser Leu Val

165 170 175

Gln Asn Glu Arg Asn Arg Gln Met Asn Gln Glu Gln Gly Leu Ala Asp

180 185 190

Lys Ile Arg Val Phe Asp Gly Ser Phe Glu Asp Leu Pro Phe Glu Glu

195 200 205

Asn Ser Tyr Asp Val Val Trp Ser Gln Asp Ala Ile Leu His Ser Gly

210 215 220

Asn Arg Arg Lys Val Leu Glu Glu Val Asp Arg Thr Leu Lys Ser Gly

225 230 235 240

Ser Asp Phe Val Phe Thr Asp Pro Met Gln Thr Asp Asn Cys Pro Glu

245 250 255

Gly Val Leu Glu Pro Val Leu Ala Arg Ile His Leu Asp Ser Leu Gly

260 265 270

Ser Val Ser Phe Tyr Arg Gln Val Gly Glu Glu Leu Gly Trp Gln Phe

275 280 285

Val Glu Phe Asp Glu Gln Thr His His Leu Val Asn His Tyr Ser Arg

290 295 300

Val Leu Gln Glu Leu Glu Ala His Tyr Glu Gln Leu Gln Pro Glu Cys

305 310 315 320

Ser Lys Glu Tyr Leu Asp Arg Met Lys Val Gly Leu Asn His Trp Ile

325 330 335

Asn Ala Gly Lys Ser Gly Tyr Met Ala Trp Gly Ile Leu Lys Phe His

340 345 350

Lys Pro

Claims (2)

1. the application of methyl transferase gene in plant stress-resistance in the cyanobacteria trimethyl-glycine route of synthesis; It is characterized in that: said gene is ApGSMT2d and ApDMT2d, is codon replacement ApGSMT2 and low codon, the synthetic acquisition of ApDMT2 frequency of occurrences in higher plant with the cereal plants preference; Wherein: the nucleotide sequence of said ApGSMT2d is shown in SEQ ID NO.9; The nucleotide sequence of said ApDMT2d is shown in SEQ ID NO.10; Said ApGSMT2 and ApDMT2 are meant glycocoll sarkosine N-methyl transferase gene ApGSMT2 and the Gene A pDMT2 of N-methylsarcosine N-methyltransgerase in the glycinebetaine route of synthesis that from cyanobacteria Aphanothece halophytica, clones; Wherein the nucleotide sequence of ApGSMT2 is shown in SEQ ID NO.5, and the nucleotide sequence of ApDMT2 is shown in SEQ ID NO.7; The method of said application is to adopt transgenic technology with producing transfer-gen plant in ApGSMT2d and the ApDMT2d gene transfered plant cell.

2. the application of methyl transferase gene in plant stress-resistance in the cyanobacteria trimethyl-glycine route of synthesis according to claim 1, it is characterized in that: said cereal plants is a corn.

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